EA045010B1 - NEW PEPTIDES AND PEPTIDES COMBINATIONS FOR USE IN IMMUNOTHERAPY OF LUNG CANCER, INCLUDING NON-SMALL CELL LUNG CANCER (NSCLC), SMALL CELL LUNG CANCER (SCLC) AND OTHER TYPES OF CANCER - Google Patents

Description

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to cancer immunotherapy. The present invention further relates to tumor-associated peptide epitopes of T cells, alone or in combination with other tumor-associated peptides, which can, for example, serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or stimulate T cells ex vivo with their transfer to the patient's body. Peptides bound to major histocompatibility complex (MHC) molecules, or peptides alone, may also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

The present invention relates to several novel peptide sequences and variants thereof derived from HLA class I molecules of human tumor cells that can be used in vaccine compositions to induce antitumor immune responses or as targets for the development of pharmaceutically/immunologically active compounds and cells.

State of the art

Lung cancer is the most common cause of cancer death among both men and women. Lung cancer is the most common cancer in the world, both in terms of incidence and mortality. In 2012, there were more than 1.8 million new cases (13% of total cancer cases) and 1.6 million deaths (20% of total cancer deaths) due to lung cancer. Lung cancer is the leading cause of cancer death among men in 87 countries and among women in 26 countries. More than one third of all newly diagnosed cases are in China. The highest rates are recorded in North America, Europe and East Asia (World Cancer Report, 2014).

Since 1987, more women have died each year from lung cancer than from breast cancer. The number of deaths among men decreased significantly between 1991 and 2003, by approximately 1.9% annually. Lung cancer mortality among women has remained stagnant after rising for several decades. These trends in lung cancer mortality reflect a decline in smoking rates over the past 30 years.

According to the National Cancer Institute (NCI), there were approximately 230,000 new cases of lung cancer and 160,000 deaths from lung cancer in the United States in 2013.

Historically, small cell carcinoma (SCLC) of the lung has been distinguished from non-small cell lung carcinoma (NSCLC), which includes histologic types: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. However, over the past decade, the distinction between adenocarcinoma and squamous cell carcinoma has become increasingly recognized due to significant differences in genetic properties as well as responses to specific therapies. Based on this, lung cancers are increasingly being classified into molecular subtypes based on specific genetic alterations that initiate and maintain tumor development in the lung (Travis et al., 2013).

The prognosis is usually disappointing. Of all patients diagnosed with lung cancer, 10-15% survive for 5 years. The poor survival rates of patients with lung cancer are, at least in part, a consequence of the fact that 80% of patients have metastases at diagnosis, and more than half of patients have distant metastases (SEER Stat facts, 2014). At the time of detection, stage IV disease is observed in 30-40% of all NSCLC cases and 60% of SCLC.

The one-year relative survival rate for patients with lung cancer has increased slightly from 35% in 1975-1979. to 44% in 2010, largely due to improvements in surgical techniques and combination treatments. However, the 5-year survival rate for all stages overall is only 17%. The survival rate for all cases detected while the disease is still localized is 54%; however, only 16% of all lung cancers are diagnosed at this early stage. (SEER Stat facts, 2014).

Treatment is determined by the type (small cell or non-small cell) and stage of cancer and includes surgery, radiation therapy, chemotherapy, and biologic targeted therapy such as bevacizumab (AVASTIN®) and erlotinib (TARCEVA®). For localized cancer, surgery is usually the treatment of choice. Recent research indicates that survival for early-stage non-small cell lung cancer improves when surgery is followed by chemotherapy. Because the disease is usually already advanced when it is discovered, radiation therapy and chemotherapy are often used, sometimes in combination with surgery. Chemotherapy alone or in combination with radiation therapy is the standard treatment chosen for small cell lung cancer; With this treatment regimen, a large percentage of patients experience remission, which in some cases is long-lasting after surgery (S3-Leitlinie Lungenkarzinom, 2011).

Advanced lung cancer was also resistant to traditional chemotherapy. However

- 1,045,010 less, recent advances have led to impressive advances in treatments based on histology and genetics. The level of scrutiny is reflected in clinical trials of adjuvant chemotherapy designed to distinguish between not only mutations in codons 12 and 13 of the KRAS gene, but also various amino acid substitutions as defined by specific mutations in codon 12 (Shepherd et al., 2013).

In order to expand the number of possible treatments for NSCLC, various immunotherapeutic approaches have been or continue to be studied. While vaccination with LBLP25 or MAGEA3 in patients with NSCLC has failed to demonstrate a survival benefit when administered, one allogeneic cell line vaccine has shown promising results in clinical trials. In addition, clinical trials of vaccines targeting gangliosides, epidermal growth factor receptor, and several other antigens are currently underway. An alternative strategy to enhance a patient's antitumor T cell response is to block inhibitory T cell receptors or their ligands with specific antibodies. The therapeutic potential of several of these antibodies, including ipilimumab, nivolumab, pembrolizumab, MPDL3280A, and MEDI-4736, in NSCLC are currently being evaluated in clinical trials (Reinmuth et al., 2015).

Considering the serious side effects and high costs associated with cancer treatment, there is a need to identify factors that can be used to treat cancer in general and lung cancer (including NSCLC (non-small cell lung cancer) and SCLC (small cell lung cancer)) in in particular. There is also a need to identify factors that constitute biomarkers of cancer in general and lung cancer (including NSCLC and SCLC) in particular, which will allow better diagnosis, prognosis and prediction of treatment success.

Cancer immunotherapy is an option to specifically target cancer cells while minimizing side effects. The existence of tumor-associated antigens is used in cancer immunotherapy.

The current classification of tumor-associated antigens (TAA) includes the following main groups.

a) Cancer-testis antigens: the first ever identified TAAs that can be recognized by T cells belong to this class, originally called cancer-testis antigens (CT), since its members are expressed in human tumors of different histological structure, and among normal tissues - only in spermatocytes/spermatogonia of the testis and occasionally in the placenta. Since testicular cells do not express HLA class I and II molecules, these antigens cannot be recognized by T cells in normal tissues and therefore can be considered immunologically tumor-specific. Well-known examples of CT antigens are members of the MAGE family and NY-ESO-1.

b) Differentiation antigens: These TAAs are found in tumor and normal tissues from which the tumor is formed. Most of the known differentiation antigens are found in melanomas and normal melanocytes. Many of these melanocyte lineage-specific proteins are involved in melanin biosynthesis and are therefore not tumor specific; however, despite this, they are widely used in anticancer therapy. Examples include, but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma or PSA for prostate cancer.

c) Overexpressed TAAs: Genes encoding widely expressed TAAs have been found in tumors of varying histological structure, as well as in many normal tissues, mostly with lower expression levels. It is possible that many epitopes processed and potentially presented by normal tissues are below the threshold level for recognition by T cells, while their overexpression in tumor cells may initiate an anticancer response by breaking previously established tolerance. Well-known examples of TAAs of this class are Her-2/neu, survivin, telomerase or WT1.

d) Tumor-specific antigens: these unique TAAs are formed as a result of mutations of normal genes (such as β-catenin, CDK4, etc.). Some of these molecular changes are associated with neoplastic transformation and/or progression. Tumor-specific antigens are generally capable of inducing strong immune responses without carrying the risk of autoimmune reactions to normal tissues. On the other hand, TAA data are in most cases only suitable for the specific tumor on which they were identified and are usually not general to many individual tumors. Tumor specificity (or association) of a peptide can also arise if the peptide is derived from a tumor (tumor-associated) exon in the case of proteins with tumor-specific (-associated) isoforms.

e) TAAs resulting from aberrant post-translational modifications: Such TAAs may arise from proteins that are neither specific nor overexpressed in tumors, but nevertheless become tumor-associated through post-translational processes that occur predominantly in tumors. Examples for this class arise from changes in the nature of glycosylation, leading to the appearance of new epi

- 2 045010 tops in tumors, as in the case of MUC1, or in events such as protein splicing during degradation, which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins and can play a leading role in the oncogenic process, and since they are foreign (not of human origin), they can provoke a T-cell response. Examples of such proteins are the viral proteins of human papillomavirus type 16, E6 and E7, which are expressed in cervical carcinoma.

The targets of T cell-based immunotherapy are peptide epitopes derived from tumor-associated or tumor-specific proteins that are presented by human major histocompatibility complex (MHC) molecules. Antigens that are recognized by tumor-specific T lymphocytes, that is, their epitopes, can be molecules formed from any class of proteins, such as enzymes, receptors, transcription factors, etc., that are expressed and, in comparison with unchanged cells of the same origin, usually have elevated levels in the cells of the corresponding tumor.

There are two classes of MHC molecules, MHC class I and MHC class II. MHC class I molecules consist of an alpha heavy chain and beta-2-microglobulin, MHC class II molecules consist of an alpha and beta chain. Their three-dimensional conformation forms a binding groove that is used for non-covalent interaction with peptides.

MHC class I molecules are found on most cells that have a nucleus. They present peptides formed by proteolytic cleavage of predominantly endogenous proteins, defective ribosomal products (DRIPs) and larger peptides. However, peptides derived from endosomal compartments or exogenous sources are also frequently found on MHC class I molecules. This non-classical method of presentation by class I is called cross-presentation in the literature. (Brossart and Bevan, 1997; Rock et al., 1990). MHC class II molecules may occur predominantly on professional antigen presenting cells (APCs) and primarily present peptides of exogenous or transmembrane proteins, which are taken up by APCs, for example, during endocytosis and subsequently processed. Peptide-MHC class I complexes are recognized by CD8-positive T cells bearing the appropriate T-cell receptor (TCR), whereas peptide-MHC class II complexes are recognized by CD4-positive helper T cells bearing the appropriate TCR. It is well known that TCR, peptide and MHC occur in a stoichiometric ratio of 1:1:1.

CD4-positive helper T cells play an important role in inducing and maintaining effective CD8-positive cytotoxic T cell responses. The identification of CD4-positive T cell epitopes derived from tumor-associated antigens (TAAs) may be extremely important for the development of pharmaceuticals to initiate antitumor immune responses (Gnjatic et al., 2003). At the tumor site, T helper cells maintain a cytokine environment favorable for CTLs (Mortara et al., 2006) and attract effector cells, for example, CTLs, natural killer (NK) cells, macrophages, granulocytes (Hwang et al., 2007) .

In the absence of inflammation, the expression of MHC class II molecules is predominantly limited to cells of the immune system, especially professional antigen-presenting cells (APCs), such as monocytes, monocyte-derived cells, macrophages, and dendritic cells. Tumor cells from cancer patients have been found to express MHC class II molecules (Dengjel et al., 2006).

The extended (longer) peptides of the invention can act as active MHC class II epitopes.

T helper cells activated by MHC class II epitopes play an important role in controlling the effector function of CTLs in antitumor immunity. T helper cell epitopes that initiate TH1 type T helper cell responses support the effector functions of CD8-positive killer T cells, which include cytotoxic functions directed against tumor cells exhibiting tumor-associated peptide/MHC complexes on their cell surface. Thus, tumor-associated T helper cell peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients in vaccine compositions that stimulate antitumor immune responses.

In mammalian animal models such as mice, it has been shown that even in the absence of CD8-positive T cells, CD4-positive T cells are sufficient to attenuate the clinical manifestations of tumors by inhibiting angiogenesis through the secretion of interferon-gamma (IFN-gamma). (Beatty and Paterson, 2001; Mumberg et al., 1999). There is evidence that CD4 T cells are direct antitumor effector cells (Braumuller et al., 2013; Tran et al., 2014).

Since constitutive expression of HLA class II molecules is usually limited to immune cells, the isolation of class II peptides directly from primary tumors was previously considered impossible.

- 3 045010 possible. However, Dengjel et al were able to identify a number of MHC class II epitopes directly from tumors (WO 2007/028574, EP 1 760 088 b1).

Since both types of responses, dependent on CD8 and CD4, contribute to the antitumor effect jointly and synergistically, the identification and characterization of tumor-associated antigens recognized by both CD8+ T cells (ligand: MHC class I molecule + peptide epitope) and CD4-positive helper T cells (ligand: MHC class II molecule + peptide epitope) are important in the development of antitumor vaccines.

In order for an MHC class I peptide to initiate (cause) a cellular immune response, it must also bind to an MHC molecule. This process depends on the allele of the MHC molecule and specific polymorphisms in the amino acid sequence of the peptide. MHC class I binding peptides are typically 8-12 amino acid residues in length and typically contain two conserved residues (anchors) in their sequence that interact with the corresponding binding groove of the MHC molecule. Thus, each MHC allele has a binding motif that determines which peptides can specifically bind to the binding groove.

In an MHC class I-dependent immune response, peptides not only must be able to bind to specific MHC class I molecules expressed by tumor cells, but they must also then be recognized by T cells bearing specific T cell receptors (TCRs).

In order for proteins to be recognized by T lymphocytes as tumor-specific or tumor-associated antigens and for them to be used in therapy, prerequisites must be met. The antigen should be expressed predominantly by tumor cells and not expressed or expressed in relatively small amounts by healthy tissues. In a preferred embodiment, the peptide should be over-presented by tumor cells compared to normal healthy tissues. In addition, it is desirable that the corresponding antigen not only be present in some type of tumor, but also have a high concentration (ie, several copies of the corresponding peptide per cell).

Tumor-specific and tumor-associated antigens are often derived from proteins directly involved in the transformation of a normal cell into a tumor cell, due to their function, for example, in cell cycle control or suppression of apoptosis. In addition, downstream targets of proteins that directly cause transformation may be overrepresented and thus be indirectly tumor-associated. Such indirect tumor-associated antigens may also be targets of vaccination approaches (Singh-Jasuja et al., 2004). It is necessary for epitopes to be present in the amino acid sequence of an antigen to ensure that such a peptide (immunogenic peptide) derived from a tumor-associated antigen leads to a T cell response in vitro or in vivo.

In essence, any peptide capable of binding to an MHC molecule can function as a T cell epitope. A prerequisite for inducing a T cell response in vitro or in vivo is the presence of a T cell with the appropriate TCR and the absence of immunological tolerance to that particular epitope.

Therefore, TAA antigens are the starting point for the development of T cell-based therapies, including, but not limited to, cancer vaccines. Methods for identifying and characterizing TAAs are usually based on the use of T cells that can be isolated from patients or healthy subjects, or they can be based on the generation of different transcriptional profiles or different peptide expression patterns between tumor and normal tissues. However, the identification of genes that are overexpressed in tumor tissues or human tumor cell lines, or that are selectively expressed in such tissues or cell lines, does not provide precise information about the use of antigens transcribed from these genes in immunotherapy. This is because only a subpopulation of the epitopes of these antigens is suitable for such use, since a T cell with the appropriate TCR must be present and there must be no or minimal immunological tolerance to that particular epitope. Therefore, in the most preferred embodiment of the invention, it is important to select only those peptides presented in excess or selectively against which a functional and/or proliferative T cell can be detected. Such a functional T cell is defined as a T cell that, when stimulated with a specific antigen, can be expanded through cloning and is capable of performing effector functions (effector T cell).

When targeting peptide-MHC complexes with specific TCRs (eg, soluble TCRs) and antibodies or other molecules that bind thereto (scaffolds) in accordance with the invention, the immunogenicity of the underlying peptides is secondary. In such cases, presentation is the determining factor.

Summary of the invention

In a first aspect, the present invention provides a peptide comprising an amino acid sequence selected from the group consisting of sequences having SEQ ID No. 1 to SEQ ID No. 489, or a variant thereof, which is at least 77%, preferably at least 88%

- 4 045010 is homologous (preferably at least 77% or at least 88% identical) to the sequence of SEQ ID No. 1 to SEQ ID No. 489, wherein said variant binds to MHC and/or induces a T cell cross-reaction with said peptide, or a pharmaceutically acceptable salt thereof, wherein said peptide is not a full-length core polypeptide.

The present invention further relates to a peptide of the present invention comprising a sequence that is selected from the group consisting of sequences having SEQ ID No. 1 of SEQ ID NO 489, or a variant thereof that is at least 77%, preferably at least 88% homologous (preferably at least 77% or at least 88% identical) to the sequence of SEQ ID No. 1 according to SEQ ID NO 489, wherein the peptide or variant thereof has a total length of 8-100, preferably 8-30 and most preferably 8-14 amino acids.

The following tables present the peptides of the present invention and their corresponding SEQ ID NO. and potential parent (underlying) genes for these peptides. In table 1 peptides with sequences with SEQ ID No. 1 to SEQ ID No. 83 bind to HLA-A*24, peptides with sequences with SEQ ID No. 84 to SEQ ID No. 133 bind to HLA-A*02, peptides with sequences with SEQ ID No. 134 to SEQ ID No. 201 bind to HLA-A*01, peptides with sequences with SEQ ID No. 202 to SEQ ID No. 219 bind to HLA-A*03, peptides with sequences with SEQ ID No. 220 to SEQ ID No. 295 bind to HLA-B*07, peptides with sequences with SEQ ID No. 296 to SEQ ID No. 318 bind to HLA-B*O8, peptides with sequences with SEQ ID No. 319 to SEQ ID No. 374 bind to HLA-B*44. Peptides from the table. 2 have been previously disclosed in extensive listings as results from high-throughput screens with high error rates or have been calculated using algorithms, but have not previously been associated in any way with cancer. In table 2 peptides with sequences with SEQ ID No. 375 to SEQ ID No. 387 bind to HLA-A*24, peptides with sequences with SEQ ID No. 388 to SEQ ID No. 393 bind to HLA-A*02, peptides with sequences with SEQ ID No. 394 to SEQ ID No. 452 bind to HLA-A*01, peptides with sequences with SEQ ID No. 453 to SEQ ID No. 458 bind to HLA-A*03, peptides with sequences with SEQ ID No. 459 to SEQ ID No. 475 bind to HLA-B*07, peptides with SEQ ID No. 476 to SEQ ID No. 489 bind to HLA-B*44. Peptides from the table. 3 are additional peptides that may be useful in combination with other peptides of the invention. In table 3 peptides with sequences with SEQ ID No. 490 to SEQ ID No. 508 bind to HLA-A*24, peptides with sequences with SEQ ID No. 509 to SEQ ID No. 528 bind to HLA-A*02, peptides with sequences with SEQ ID No. 529 to SEQ ID No. 530 binds to HLA-B*07, peptide with SEQ ID No. 531 binds to HLA-B*44.

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Table 1

Peptides according to the present invention

Seq ID No Subsequence Gene(s) Allotype HI_A 1 QYDPTPLTW ADAMTS12 A*24 2 VWSNVTPLKF MMP12 A*24 3 YLEKFYGL MMP12 A*24 4 SYEKVINYL MAGEA9, MAGEA9B A*24 5 RYMKKDYLI SLC35D3 A*24 6 KYKDYFPVI MAGEC2, LOC392555 A*24 7 VQQWSVAVF PTHLH A*24/B*15 8 PFLPPAACFF ASCL1 A*24 9 RILRFPWQL MMP11 A*24/A*32 10 VWSDVTPLNF MMP13 A*24 eleven YYSKSVGFMQW FAM111B A*24 12 STIRGELFFF MMP11 A*24/B*57 13 HYTYILEVF SLC7A11 A*24 14 SYSSCYSF KRT13, KRT17 A*24 15 KYALLLQDL PLEKHG4B A*24 16 TYNPDFSSL SP9 A*24 17 YYADKKTFIVL SCN9A A*24 18 DYIGSVEKW HS6ST2 A*24 19 ILKEDPFLF MACC1 A*24 20 EFTTVLYNF TP63 A*24 21 SYEVRSTF TAS2R38 A*24 22 TQPGDWTLF POSTN A*24/B*15 23 KFIISDWRF FAM83A A*24 24 MYPDLSELLM NUP155 A*24 25 SYNGYVFYL ROS1 A*24 26 KTPTNYYLF NMUR2 A*24/B*35 27 NYTLYPITF GXYLT1 A*24 28 YYSIISHTL ROS1 A*24 29 VYPLLSRLYW ROS1 A*24 thirty QYLPGWTVLF SLC22A31 A*24 31 QYQNVLTLW DST A*24 32 SLPDLTPTF LAMB3 A*24 33 KSSVIASLLF TMTC3 A*24/B*57 34 MQPRMFFLF FGD6 A*24 35 KYLEESVWL GPR98 A*24 36 KQMEDGHTLF UHRF1 A*24/B*15 37 QWPWQASLQF TMPRSS3 A*24 38 KYTNWKAFL MBTD1 A*24 39 LIFMLANVF GABRP A*24/A*32 40 QYEPPSAPSTTF DROSHA A*24 41 VIYFMGAIF OR7E24 A*24/B*15

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42 TLPNTIYRF ROS1 A*24 43 IQMDEPMAF CDC7 A*24/B*15 44 AYLSAVGTF ABCC1 A*24 45 KYFVPPQLF B3GNT6 A*24 46 AFPVTSIFHTF KCNG1, KCNG2 A*24 47 KYADYFLEV CCNJ A*24 48 VFIDHPVHLKF TENM4 A*24 49 LYISEVRNI DST A*24 50 SYPELVKMVW C5orf34 A*24 51 KYALLLQEL PLEKHG4 A*24 52 KYMKIFHKF ZNF681 A*24 53 KYITNLEDL TXNDC16 A*24 54 LLIKLLQTF PRKDC A*24/B*15 55 RWMDQRLVF GABRP A*24 56 VYMIEPLEL ADAM23 A*24 57 YPSIIQEF POLA1 A*24 58 QFAAPLRGIYF C1QTNF6 A*24 59 KYSTTFFMV XPR1 A*24 60 TYLSIFDQL SF3A3 A*24 61 NYAENILTL FIGNL1 A*24 62 LYQEILAQL URB1 A*24 63 VMPSDSFFF GAL3ST4 A*24 64 NYAIFDEGHML SMARCAD1 A*24 65 VYPASKMFPFI CKAP5 A*24 66 IYFRDSSFL TEP1 A*24 67 RYPGKFYRV ZAK A*24 68 IYQQIIQTY NCAPG2 A*24 69 IMPEKFEFW CHD2 A*24 70 PYTNYTFDF CNOT6, CNOT6L, CNOT6LP1 A*24 71 SYMVLAPVF SPIN1, SPNS1 A*24 72 RYEGILYTI LSM14A, LSM14B A*24 73 SYIGLPLTL NPC1 A*24 74 VYDQYFITL ATP8B2 A*24 75 NYIYSISVF PLA A A*24 76 WYGWHFPEL NOP58 A*24 77 AYTLLGHEFV CDC27 A*24 78 TWFPKTPMLF KBTBD2 A*24 79 RYLADLPTL CEP85 A*24 80 YYSPLRDLL Rasa3 A*24 81 LYPEGLRLL ATP6V0D2 A*24 82 RFLPSPVVI CUL3 A*24

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83 TYCQNIKEF EIF3H A*24 84 YVDINTFRL MMP12 A*02 85 YIDEFQSLV RTL1 A*02 86 FVIDGFDEL NLRP2 A*02 87 TLYPYQISQL KIF26B A*02 88 VQMVITEAQKV LAMC2 A*02 89 ILSTTMVTV KIF26B A*02 90 FLLMHPSI NDST4 A*02 91 FALPGLLHA LAMB3 A*02 92 NLRDLLSEV KIF26B A*02 93 TLQEKILQV MYO3A A*02 94 VLPDIETLIGV TET1 A*02 95 ITIGVLARV CEACAM6 A*02 96 HLVGGLHTV DACH1, DACH2 A*02 97 VLALVNSTV CBFA2T2 A*02 98 LQSSGLTLLL MSLNL A*02/B*13 99 FLKEKVPGI CDKAL1 A*02 100 RQYPTPFQL ZNF280C A*02/B*48 101 FIISDWRFVL FAM83A A*02 102 SLLEQAIAL ST18 A*02 103 FLYYPDPVL PLXNA1 A*02 104 GMLDIFWGV RASSF6 A*02 105 SLLTHIPTA PLEKHG4 A*02 106 FIIDTTYPAYV FAP A*02 107 LLQGAIESV PLEKHG4 A*02 108 MIIALSLY GPR33 A*02 109 LLLGSIGLLGV OPN3 A*02 110 LLADFQALL CCDC87 A*02 111 ALCLLLHLL MRGPRE A*02 112 SVSDGIHSV CELSR1 A*02 113 AVLTGLVEV LRBA A*02 114 ILDERQVLL ITGAE A*02 115 MLLETQDALYV ADORA2B A*02 116 VLMEENSKL HAP1 A*02 117 FLDPNARPLV CAD A*02 118 ALSSVLHSI FGD6 A*02 119 RTADITVTV ITGAE A*02 120 ALLANLPAV SLC26A9 A*02 121 ALVDTLTGI IPO9 A*02 122 ALLEMFPEITV TXNDC16 A*02 123 LMAFFLAVV OR6C75 A*02 124 SVASVLLYL PRKDC A*02

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125 VLQPFLPSI IPO9 A*02 126 FLSTVTSV SOGA2 A*02 127 GLDGSLVFL MARCH6 A*02 128 FLGTTPTL SF3B3 A*02 129 VLYDKDAVYV BMS1 A*02 130 NLWGGQGLLGV GORASP2 A*02 131 LLKEFVQRV COL6A3 A*02 132 ALWLVDPLTV SLC33A1 A*02 133 MTLPVDAVISV UFSP2 A*02 134 AAEIGDKSWLY PLA2G7 A*01 135 ASEDSVLLY CHAF1B A*01 136 ATDLVVLDRY DICER1 A*01 137 ATSKFMEFY EDNRA A*01 138 DSDSCHFNY DCBLD2 A*01 139 ECDMAFHIY UHRF1, LOC728688 A*01 140 ESDREELNY CBFA2T2 A*01 141 ESDVGWVY DDX60L A*01 142 EVAEPSVLFDLY C9orf114 A*01 143 FIDYPKKEDY PLAU A*01 144 FLDSQNLSAY BBS1, DPP3 A*01 145 FVDKPVAY TAF1B A*01 146 GLNTGSALSY COL6A3 A*01/B*15 147 GSSDSSTLPKL TDRD5 A*01 148 GTEFTTILY TP73 A*01 149 GTEFTTVLY TP63 A*01 150 GTELLSLVY PRKDC A*01 151 HSDLKVGEY DICER1 A*01 152 HTDSLHLLI ANKRD52,FLJ25613 A*01 153 KLDRSVFTAY FAM111B A*01 154 LLDISQKNLY ZNF439 A*01 155 LLDPNPHMY RGS17 A*01 156 LLDSLREQY SESTD1 A*01 157 LMDRPIFY GALNS A*01 158 LSDLLKQGY PKD2L1 A*01 159 LSDTSVIQFY C16orf62 A*01 160 LTEAVLNRY KIF26B A*01 161 LVDDGTHGQY TRPM2 A*01 162 LVDNSIRELQY CARD8 A*01 163 NSDSSLTLREFY FSTL4 A*01 164 NTDNLAVY CXorf61 A*01 165 NTDPTAPPY CDH3 A*01 166 NTQITDIGRY HMCN1 A*01

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167 QSDPGTSVLGY SEZ6 A*01 168 QTDHPQPILDRY ITGAE A*01 169 RLDTPLYFSY LEPRE1 A*01 170 RSDDTAVYY IGHA1, IGHA2, IGHD, IGHG3, IGHV1-18, IGHV1-2, IGHV4-31 A*01 171 RSDPVTLNVLY CEACAM6, PSG1, PSG2, PSG4, PSG5, PSG7, PSG8 A*01 172 RTDSCSSAQAQY DCBLD2 A*01 173 RTEFNLNQY COL12A1 A*01 174 SADDIRGIQSLY MMP12 A*01 175 SDVTPLTF MMP11 A*01 176 SRTINVSNLY LAMA1 A*01 177 SSDEVNFLVY TBL1XR1 A*01 178 SSDSSTLPKL TDRD5 A*01/C*12 179 STAKSATWTY TP63, TP73 A*01 180 STDPWIQMAY KDM1B A*01 181 TADGKTYYY TCERG1 A*01 182 TDYHVRVY FNDC3B A*01 183 TLEDIATSHLY CBFA2T2 A*01 184 TSAHPEDSSFY LOC731467, TRBV20-1 A*01 185 TSDSNLNKY KCNH7 A*01 186 TTDIIEKY DDX60L A*01 187 VADLHLY GARS A*01 188 VSDAKLDKY RCOR2, LOC441644 A*01 189 VSDSECLSRY LAMA1 A*01 190 VTDGINPLIDRY FREM2 A*01 191 VTDGSLYEGVAY DSE A*01 192 VTEESFDSKFY CDKAL1 A*01 193 VTEFSLNTY COL6A3 A*01 194 VVADTKMIEY ADAMTS12 A*01 195 VVDSVGGYLY ROS1 A*01 196 WMFFVINY TMEM217 A*01 197 YADTVRPEFY COL6A3 A*01 198 YLDPVQRDLY ZNF655 A*01 199 YLPQHTIETY TP63 A*01/B*15 200 YSDEDVTKY SDK2 A*01 201 YVGKEHMFY MAGEA9, MAGEA9B A*01 202 KLAELEGALQK KRT81, KRT121P, KRT83, KRT85, KRT86 A*03 203 KVKDTPGLGK KIF26B A*03 204 AVFDKFIRY BTBD17 A*03

- 10 045010

205 SLDGAARPK SP6 A*03 206 KLIDLSQVMY MACC1 A*03/B*15 207 RSFNGLLTMY LAMB3 A*03/B*15 208 GLASRILDAK LAMB3 A*03 209 RTQIPMSEK RASSF6 A*03 210 ATSGVPVYK SLC44A5 A*03 211 TVNPVAIHK GLI2 A*03 212 KAYEQVMHY FOXA2 A*03 213 LNINMTSPMGTK PCSK2 A*03 214 RTMSEAALVRK RASSF6 A*03 215 MMFSGPQILKL ABCC1 A*03/A*32 216 KLYAWELAF ABCC1 A*03/A*32 217 RILNQILYY FGD6 A*03 218 KTLVAELLILK POLQ A*03 219 RLRSSLVFK FAM83B A*03 220 SPSVSQLSVL PRAME B*07 221 VPDVAQFVL MMP1 B*07 222 NPFYPEVEL MMP1 B*07 223 YPKDIYSSF MMP1 B*07 224 GPQPWHAAL MMP11 B*07 225 LPFDGPGGIL MMP11 B*07 226 SPRMSGLLSQT DLL3 B*07 227 YPRGNHWAVGH GRP B*07 228 YPRGNHWAVGHL GRP B*07 229 VPLPAGGGTV GRP B*07 230 VPLPAGGGTVL GRP B*07 231 RPRALRDLQL NLRP7 B*07 232 RPRALRDLQLL NLRP7 B*07 233 KPYQGNPTF DNAH17 B*07 234 RAKNAGVTI LAMC2 B*07 235 MPLKHYLLL LRRC15 B*07 236 RVRGGEDGDRAL INSM1 B*07 237 RPAATAVISL SLC7A11 B*07 238 KPGPPWAAF DCBLD2 B*07 239 YVPSASLFML E2F7 B*07 240 SPREVTVL DCBLD2 B*07 241 SARLATDAL FAM83A B*07 242 SPRWLPVSL BTBD17 B*07 243 RPIENRILIL PSG1, PSG3, PSG4, PSG5, PSG6, PSG7, PSG8, PSG9 B*07 244 FPYVRDFVM COL6A3 B*07/B*35

- 11 045010

245 RIREHVPQL COL6A3 B*07 246 TPLPAVIVL SLC7A11 B*07 247 RALLARLLLL PLAU B*07 248 IPNWARQDL NLRP7 B*07 249 VPSSRILQL THEG B*07 250 SPRDFLSGL ABCA2, TPH1 B*07 251 VPRSSGQTV SP6 B*07 252 SPDIRNTTV DCBLD2 B*07 253 RVIDAVRFTL TP63 B*07 254 NPFPHLITL ROS1 B*07 255 MPLLENLYL MXRA5 B*07 256 SPRVPSIEL COL7A1 B*07 257 LPRIPFADV ROS1 B*07 258 LPRGPLASL CDH3 B*07 259 RPPAAGLRGISL SEZ6L B*07 260 YPQHPGLNA SOX2 B*07 261 APSARVGVC KRT86 B*07 262 SAYPQRLEI CYP24A1 B*07/B*51 263 HPAPYGDLL GLI2 B*07 264 RPILIIITL TP73 B*07 265 SPRQPPRLV CYP24A1 B*07 266 HAYPPGPGL MMP10 B*07/C*03 267 HPELVNHIVF GALNT5 B*07/B*35 268 YPLFRGINL COL5A1 B*07 269 APRAPRLML ITGA3 B*07 270 APGPRFLVT CD109 B*07 271 MPLPWSLALP EGFL6 B*07/B*35 272 MPLPWSLALPL EGFL6 B*07 273 MPLLWLRGF INHBA B*07/B*35 274 TPYQEHVAL ZNF618 B*07/B*35 275 APHPPLSVV IQGAP3 B*07 276 LPRAGGAFL LAMB3 B*07 277 MPLFEPRVF PTK7 B*07/B*35 278 HPMIDINGIIVF COL5A1 B*07/B*35 279 SPARASPAL TMPRSS13 B*07 280 VPISEEGTPVL KIAA0754 B*07 281 RPRAPVTPA HES6 B*07 282 MPQIETRVIL ECT2 B*07 283 RPHSLSSEL ITGAE B*07 284 FPVTSIFHTF KCNG1, KCNG2 B*07/B*35 285 FPSFLTNSL CEP192 B*07/B*35 286 VPTLRSEL DST, MACF1 B*07

- 12 045010

287 APREEQQRSL KIAA1211 B*07 288 FPQKFIDLL SASS6 B*07 289 VPENHSVAL FAM83B B*07 290 APYRPPDISL TANC2 B*07 291 SPQRLRGLL CTHRC1 B*07 292 SPQRLRGLLL CTHRC1 B*07 293 RPRSALPRLLLP FZD2 B*07 294 GPTPNTGAAL COL6A3 B*07 295 KPEGTRIAV COL6A3 B*07 296 MPMQDIKM PRAME B*08 297 RAQLKLVAL KLHDC7B B*08 298 FNKRKPLSL NLRP2 B*08 299 MAQFKEISL NLRP2 B*08 300 VASPKHCVL KIF26B B*08 301 YMHKLLLVL PTH2 B*08/B*35 302 HLLQKQTSI TP63 B*08 303 LPFPKFTV GALNT5 B*08 304 ELKKLYCQI TP63 B*08 305 ALKLRVAVL C16orf59 B*08 306 ILKVKVGL POSTN B*08 307 ILLPRTVSL MXRA5 B*08 308 MLKQKVEEL DST B*08 309 DAIQRKYSC DST B*08 310 LPPKKFVL NUP155 B*08 311 EIRIRWQM PRKDC B*08 312 EAMLRNKEL CENPF B*08 313 ELKKKEYEEL CENPF B*08 314 AIISRLVAL TAN 02 B*08 315 DIYQRALNL VPS13B B*08 316 VIKEKALTL USP9X, USP9Y B*08 317 LVKVKVLL ARID4A B*08 318 EAAIRSVEL DSCR3 B*08 319 AEMLERVIKNY MAGEA4 B*44 320 MEVDPIGHVYIF MAGEA3, MAGEA6 B*44 321 AEMLESVIKNY MAGEA1, MAGEA8, MAGEA9, MAGEA9B B*44 322 KEVDPAGHSY MAGEA8, MAGEA9, MAGEA9B B*44 323 SEFMQVIF MAGEA9, MAGEA9B B*44 324 TDSIHAWTF SLC35D3 B*44/B*18 325 QEQDVDLVQKY MMP1 B*44 326 QEMQHFLGL MMP12 B*44

- 13 045010

327 YEIEARNQVF MMP12 B*44/B*18 328 FEYDFLLQRI MMP12 B*44 329 NEHPSNNW LAMC2 B*44 330 KEGDLGGKQW ADAMTS12 B*44 331 EDAQGHIW MMP11 B*44 332 MEVPVIKI ECT2 B*44 333 AETLSTIQI KIF26B B*44 334 AEDEPAAAHL KIF26B B*44 335 KELEATKQY KIF26B B*44 336 ASSSGPMRWW LAMB3 B*44/B*57 337 TENRYCVQL JUP, KRT13, KRT17 B*44 338 SEGSEPALLHSW FAM83A B*44 339 SEPALLHSW FAM83A B*44 340 TEFSLNTY COL6A3 B*44 341 EEIEGKGSFTYF POSTN B*44 342 HEFSSPSHL TP63 B*44 343 TEFTTVLY TP63 B*44 344 EEATGQFHVY CEACAM1, CEACAM3, CEACAM6 B*44 345 IEFIHPQAF MXRA5 B*44 346 VEAPGPVHVYW PTK7 B*44 347 ALNPYQYQY DLX5 B*44/A*29 348 AEIQGNINHV IQGAP3 B*44 349 AEQDMRELTY DST B*44 350 GECDVFKEIL DCBLD2 B*44 351 EEVNYINTF CCNE2 B*44 352 NEVLTYIKF ABCC5 B*44 353 GEIIMQNNW SERTAD4 B*44 354 TEDPTILRI PLXNA1 B*44 355 SDMVRFHLF SGK196 B*44 356 EEGRVYLF ITGA2 B*44 357 RELENCFQIQ DNAH14 B*44 358 KEADIHFLI COL6A5 B*44 359 DELFSIALY NUP155 B*44 360 AEVPTTGVII ITGA2 B*44 361 SENLFASF ITGA2 B*44 362 SEKGVIQVY NUP155 B*44 363 AELDKLTSV CENPF B*44 364 AETPIQNVI MET B*44 365 SEMNVNMKY MET B*44 366 AENLFRAF PRKDC B*44 367 GEVHPSEMI PRKDC B*44 368 GEFPVVRVQV YEATS2 B*44 369 EEIERFFKL NUP155 B*44 370 YEDLSQKY CENPF B*44 371 GELALKKKI PRKDC B*44 372 TEGIIMKDF MET B*44 373 MEMQKSPVF FSTL4, GRM7, LOC440173, LOC644919, LOC728755, SLC44A5 B*44 374 DEVNFLVY TBL1X, TBL1XR1, TBL1Y B*44/B*18

- 14 045010

table 2

Additional peptides of the present invention whose association with cancer has not been previously known Seq ID No Subsequence Gene(s) HLA allotype 375 VYSDLHAFYY MANEAL A*24 376 KYVKDFHKF ZNF724P A*24 377 VYVGAVNRI PLXNA1 A*24 378 KFLGPAEHLTF PROM2 A*24 379 NYIVPDKQIF POLA1 A*24 380 VFQEKHHVI MOXD1 A*24 381 TYSKKHFRI CHEK2 A*24 382 IYHSHHPTL OPA1 A*24 383 RYKQDVERF SMC5 A*24 384 KYVKVFDKF ZNF107 A*24 385 MYINEVERL PTPN14 A*24 386 VYNDHSIYVW MAPKBP1 A*24 387 RWLPQKNAAQF DOCK5, PPP2R2A A*24 388 FSIPEGALVAV ABCC1 A*02 389 TLMEQPLTTL TXNDC16 A*02 390 HIMPTVHTV ADNP2 A*02 391 SLIDMRGIETV SMC6 A*02 392 SLFKDQMEL IPO8 A*02 393 ILLPYLQTL TIPAR A*02 394 ASEAEMRLFY DHX37 A*01 395 ASEASRLAHY HIST1H2BA, HIST1H2BL, HIST3H2BB A*01 396 ASEFGNHYLY SF3B3 A*01 397 ASEITSKGASLY CLUAP1 A*01 398 ASEQQALHTVQY NUP188 A*01 399 ATDIPCLLY RINT1 A*01 400 ATDISRQNEY PDE7A A*01 401 DDSDESYMEKSLY CLSPN A*01 402 DTDSQRLAY E2F1 A*01

- 15 045010

403 ELDSKVEVLTY SNX7 A*01 404 ETARKFLYY GPD2 A*01 405 ETEEGIYWRY KREMEN2 A*01 406 ETEQTKFWDY FUT11 A*01 407 FSDNDKLYLY RFX5 A*01 408 FTEQWTDGY TLK2 A*01 409 FVDPLVTNY TRIT1 A*01 410 GSDHQSPSSSSSY ZBTB43 A*01 411 GTVYEDLRY UBE2C A*01 412 ILDEVIMGY KIF11 A*01 413 ISDRYYTALY CEBPZ A*01 414 KTDESLTKY CHD8 A*01 415 LLDPRSYHTY DOCK8 A*01 416 LLDTAQKNLY ZNF614 A*01 417 LLEDKHFQSY WDR6 A*01 418 LSDPSGPKSY RPS6KC1 A*01 419 LSELKPMSY TMTC3 A*01 420 LTEDKETLQY TUBGCP2 A*01 421 LTELLERAAFY SLC15A4 A*01 422 MIDVTKSYY DCTN5 A*01 423 NLDAVHDITVAY LCLAT1 A*01 424 NLDEEKQLLY FRMD6 A*01 425 NLDIIQQEY WDR75 A*01 426 NLDQATRVAY SMC4 A*01 427 NSDEQKITEMVY LRBA A*01 428 NSELSCQLY TYMS A*01 429 NTEDSSMSGYLY FGD6 A*01 430 NTEGLHHLY SMEK2, SMEK3P A*01 431 NTSDMMGRMSY ARID1A A*01 432 NVDPVQHTY AGRN A*01 433 QIDTGENLY ZNF267 A*01 434 QTDCAPNNGY NOMO1, NOMO2, NOMO3 A*01 435 QTDDTTWRTEY ZMYM2 A*01 436 QTETGTPYMLY RRM1 A*01 437 STDGKHWWEY CCNT1, CCNT2 A*01 438 STDNFNCKY FGD6 A*01 439 TLDAGKFQIY DHX15 A*01 440 TLDENPGVRY STXBP3 A*01 441 TLDSALNAASYY TCTN3 A*01 442 TSDFSRFTNY CCNE2 A*01 443 TTDFPSESSFEY CXorf21 A*01 444 TTDTVIRSY SETD4 A*01

- 16 045010

445 VLDQGKITEY ABCB10 A*01 446 VTAQWGTERY PLD2 A*01 447 WDEDHELIY CHST11 A*01 448 YLDIPNPRY CIT A*01 449 YLDRGTGNVSFY TRIM4 A*01 450 YSDDGQKWTVY DCBLD2 A*01 451 YSDSLVQKGY MSH6 A*01 452 YVDAVLGKGHQY NUP160 A*01 453 AINTSIKNK TRPM8 A*03 454 KVYTPSISK CDKAL1 A*03 455 RIADIFVKK FGD6 A*03 456 SMFTAILKK LRBA A*03 457 SINKPTSER NDC80 A*03 458 GIADFVLKY RNFT2 A*03 459 RPMQQARAQL KLHDC7B B*07 460 MPMAGDMNGL TP63 B*07 461 RPILIIVTL TP63 B*07 462 RPFHTRATV KIF26B B*07 463 TPKAGPTL KIF26B B*07 464 YPRPGTPAA CDKAL1 B*07 465 VPRPIFSQL GREB1, GREB1L B*07 466 APYKSVTSL FGD6 B*07 467 KPFSSFTSM RASSF6 B*07 468 SPMYGQAGL FOXA2 B*07 469 YPENGVVQM UHRF1 B*07 470 SPNSYFRVL PCDHB13, PCDHB8 B*07 471 KPRPDVTNEL CDCA7 B*07 472 NPRATDAQL LRBA B*07 473 LPRALLSSL IL4I1 B*07 474 LPRLLPAL HEATR2 B*07 475 RPHKPGLYL MANEA B*07 476 AEEEEIMKKI IGF2BP3 B*44 477 QENSYQSRL LAMC2 B*44 478 SEIEQEIGSL LAMC2 B*44 479 AEIQPQTQV PTK7 B*44 480 GEVSGLTKDF CDKAL1 B*44 481 RELQHEHSL FAT1 B*44 482 TEREWADEW CBFA2T2 B*44 483 EENDQSTHKW YEATS2 B*44 484 AEVGFVRFF MSH2 B*44 485 SEIEDSTKQVF BRCA2 B*44 486 SEDDPILQI NUP155 B*44 487 AEDQLHHSF GXYLT1 B*44 488 TEFPIIKMY TXNDC16 B*44 489 SEIGKAVGF CLSPN B*44

- 17 045010

Table 3

Peptides according to the present invention useful, for example, for personalized anti-cancer therapy

Seq ID No Subsequence Gene(s) HLA allotype 490 SYVKVLHHL MAGEA12, LOC101060230 A*24 491 KYLEKYYNL MMP1 A*24 492 NYEDHFPLL MAGEA10 A*24 493 TYKYVDINTF MMP12 A*24 494 RYLEKFYGL MMP12 A*24 495 SYNDALLTF TRPM8 A*24 496 VFMKDGFFYF MMP1 A*24 497 NYPKSIHSF MMP12 A*24 498 EYIRALQQL ASCL1 A*24 499 VYFVAPAKF LAMC2 A*24 500 VWSDVTPLTF MMP11 A*24 501 GYIDNVTLI LAM C2 A*24 502 SVHKITSTF LAMC2 A*24 503 VHFEDTGKTLLF MMP13 A*24 504 VYEKNGYIYF MMP13 A*24 505 AYISGLDVF DNAH17 A*24 506 RYVFPLPYL SOX 14 A*24 507 VYIAELEKI SMC1B A*24 508 IYVTGGHLF KLHDC7B A*24 509 ALLEEEEGV MAGEA4 A*02 510 KVLEHWRV MAGEA4, MAGEA8 A*02 511 KIWEELSVLEV MAGEA3, MAGEA6 A*02 512 VLGEEQEGV MAGEA9, MAGEA9B A*02 513 KLVELEHTL CXorf61 A*02 514 VQLDSIEDLEV PRAME A*02 515 KIFEMLEGV CT45A1, CT45A2, CT45A3, CT45A4, CT45A5, CT45A6, LOC101060208, LOC101060210, LOC101060211 A*02 516 YTFSGDVQL MMP1 A*02 517 TLYNPERTITV IGF2BP1, IGF2BP3 A*02 518 GLLEDERALQL MEX3A A*02 519 KIQEILTQV IGF2BP3 A*02 520 KIQEMQHFL MMP12 A*02 521 FVYGEPREL MAGEC2, LOC392555 A*02 522 TLDEKVAEL MAGEC2 A*02 523 HLIAEIHTA PTHLH A*02 524 KVWSDVTPL MMP11, MMP13 A*02 525 RLDDLKMTV LAMC2 A*02 526 VLSPFILTL KLHDC7B A*02 527 LLDSVSRL LAMC2 A*02 528 RLLDSVSRL LAM C2 A*02 529 HPSAHDVIL LAMC2 B*07 530 APAWLRSAA MMP11 B*07 531 AEIEADRSY LAM C2 B*44

The present invention also relates generally to peptides according to the present invention for use in the treatment of proliferative diseases, for example, acute myeloid leukemia, breast cancer, bile duct cancer, brain cancer, chronic lymphocytic leukemia, colorectal carcinoma, esophageal cancer, bile duct cancer bladder, stomach cancer, squamous cell carcinoma of the head and neck, hepatocellular cancer, melanoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, bladder cancer and uterine cancer.

Particularly preferred are peptides, alone or in combination, of the present invention selected from the group consisting of the sequences of SEQ ID NO: 1 through SEQ ID NO: 489. More preferred are peptides, alone or in combination, selected from the group , consisting of sequences from SEQ ID NO: 1 to SEQ ID NO: 374 (see Table 1), and their use in immunotherapy of lung cancer (including NSCLC and SCLC), acute myeloid leukemia, breast cancer, biliary cancer duct cancer, brain cancer, chronic lymphocytic leukemia, colorectal carcinoma, esophageal cancer, gallbladder cancer, stomach cancer, flat

- 18 045010 head and neck cell carcinoma, hepatocellular carcinoma, melanoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, bladder cancer, uterine cancer and, preferably, lung cancer (including NSCLC and SCLC ).

Thus, the present invention in another aspect relates to the use of peptides in accordance with the present invention for - preferably combined - treatment of proliferative diseases selected from the group: lung cancer (including NSCLC and SCLC), acute myeloid leukemia, breast cancer, cancer bile duct, brain cancer, chronic lymphocytic leukemia, colorectal carcinoma, esophageal cancer, gallbladder cancer, gastric cancer, squamous cell carcinoma of the head and neck, hepatocellular cancer, melanoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, bladder cancer, uterine cancer.

The present invention further provides peptides of the present invention having the ability to bind to a human major histocompatibility complex (MHC) class I molecule or - in an extended form, such as the MHC class II length variant.

The present invention further relates to peptides in accordance with the present invention, wherein said peptides (each) consist or consist essentially of an amino acid sequence in accordance with SEQ ID No. 1 to SEQ ID No. 489.

The present invention further relates to peptides in accordance with the present invention, where the specified peptide is modified and/or includes non-peptide bonds.

The present invention further relates to peptides in accordance with the present invention, wherein said peptide is part of a fusion protein, in particular fused to the N-terminal amino acids of the HLA-DR antigen-associated invariant chain (li), or fused to an antibody (or inserted into the sequence) , such as, for example, an antibody specific for dendritic cells.

The present invention further relates to nucleic acid encoding peptides in accordance with the present invention. The present invention further relates to a nucleic acid according to the present invention, which is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable of expressing and/or expressing a nucleic acid in accordance with the present invention.

The present invention further relates to a peptide in accordance with the present invention, a nucleic acid in accordance with the present invention, or an expression vector in accordance with the present invention for use in the treatment of diseases and medicine, in particular in the treatment of cancer.

The present invention further relates to antibodies that are specific for the peptides of the present invention or complexes of said peptides of the present invention and MHC and methods for their preparation.

The present invention further relates to T cell receptors (TCRs), in particular to soluble TCRs and cloned TCRs embedded in autologous or allogeneic T cells, and methods for their production, as well as natural killer (NK) cells or other cells, carrying the specified TCR or cross-reacting with the specified TCR.

Antibodies and TCRs are additional embodiments of the immunotherapeutic use of peptides in accordance with the present invention.

The present invention further relates to a host cell comprising a nucleic acid in accordance with the present invention or an expression vector as previously described. The present invention further relates to a host cell according to the present invention, which is an antigen presenting cell, preferably a dendritic cell.

The present invention further relates to a method for producing a peptide in accordance with the present invention, the method comprising culturing a host cell in accordance with the present invention and isolating the peptide from said host cell or its culture medium.

The present invention further relates to said method in accordance with the present invention, wherein the antigen is loaded onto MHC class I or II molecules expressed on the surface of a suitable antigen presenting cell or artificial antigen presenting cell upon contact of a sufficient amount of the antigen with the antigen presenting cell.

The present invention further relates to a method in accordance with the present invention, wherein the antigen presenting cell includes an expression vector capable of expressing or expressing the specified peptide containing the sequence of SEQ ID NO. 1 to SEQ ID NO. 489, preferably containing SEQ ID NO. 1 to SEQ ID NO. 374 or its variant amino acid sequence.

The present invention further relates to activated T cells produced by a method in accordance with the present invention, wherein said T cell selectively recognizes a cell that expresses a polypeptide comprising an amino acid sequence in accordance with the present invention.

The present invention further relates to a method of killing target cells in a patient whose

- 19 045010 target cells aberrantly express a polypeptide comprising any amino acid sequence in accordance with the present invention, the method comprising administering to the patient an effective number of T cells obtained in accordance with the present invention.

The present invention further relates to the use of any described peptide, nucleic acid in accordance with the present invention, expression vector in accordance with the present invention, cell in accordance with the present invention, activated T lymphocyte, T cell receptor or antibody or other molecules that bind to the peptide and /or a peptide-MHC complex according to the present invention as a medicament or in the manufacture of a medicament. Preferably, said medication has an active anticancer effect.

Preferably, the medicament is intended for cell therapy, is a vaccine or a soluble TCR protein or antibody.

The present invention further relates to use in accordance with the present invention, wherein said cancer cells are lung cancer cells (including NSCLC and SCLC), acute myeloid leukemia, breast cancer, bile duct cancer, brain cancer, chronic lymphocytic leukemia, colorectal carcinoma, esophageal cancer, gallbladder cancer, gastric cancer, squamous cell carcinoma of the head and neck, hepatocellular cancer, melanoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, bladder cancer, uterine cancer and, preferably, lung cancer (including NSCLC and SCLC).

The present invention further relates to biomarkers based on the peptides in accordance with the present invention, in the context of the invention called targets, which can be used in making a diagnosis of cancer, preferably lung cancer (including NSCLC and SCLC). The marker may be overpresentation of the peptide(s) itself or overexpression of the corresponding gene(s). These markers can also be used to predict the likelihood of success of treatment, preferably immunotherapy, and most preferably immunotherapy directed at the same target that has been identified by the biomarker. For example, an antibody or soluble TCR can be used to stain tumor sections to detect the presence of a peptide of interest complexed with MHC.

Optionally, the antibody has an additional effector function, such as an immunostimulatory domain or a toxin.

The present invention also relates to the use of these new targets in the context of cancer treatment.

Stimulation of immune responses depends on the presence of antigens that are recognized by the host immune system as foreign. The discovery of the existence of tumor-associated antigens has raised the possibility of using the host immune system to interfere with tumor growth. Various mechanisms controlling both branches of the immune system, both humoral and cellular, are currently being explored for cancer immunotherapy.

Specific elements of cellular immune responses are capable of specifically recognizing and destroying tumor cells. Isolation of T cells from populations of tumor-infiltrating cells or from peripheral blood suggests that such cells play an important role in the natural immune defense against cancer. CD8-positive T cells, which recognize peptides bound to major histocompatibility complex (MHC) class I molecules, play an important role in this response. These peptides typically consist of 8–10 amino acid residues derived from proteins or defective ribosomal products (DRIPs) found in the cytosol. Human MHC molecules are also called human leukocyte antigens (HLA).

All terms used herein, unless otherwise specified, have the meanings given below.

The term T cell response refers to the specific proliferation and activation of effector functions induced by a peptide in vitro or in vivo. For MHC class I-restricted cytotoxic T cells, effector functions may include lysis of target cells loaded with the peptide, loaded with a peptide precursor, or target cells naturally presenting the peptide; secretion of cytokines, preferably interferon-gamma, TNF-alpha or IL-2, induced by the peptide; peptide-induced secretion of effector molecules, preferably granzymes or perforins, or degranulation.

The term peptide as used herein refers to a series of amino acid residues linked to each other, usually by peptide bonds between the alpha-amine and carbonyl groups of adjacent amino acids. The peptides preferably have a length of 9 amino acids, but can be shorter - 8 amino acids in length, and longer - 10, 11 or 12 or longer and in the case of peptides associated with MHC class II molecules (extended versions of the peptides of the invention), they can have length of 13, 14, 15, 16, 17, 18, 19 or 20 or more amino acids.

In addition, the concept of peptide includes salts of a series of amino acid residues linked to each other, usually by peptide bonds between the alpha-amine and carbonyl groups of adjacent amino acids. Preferably, the salts are pharmaceutically acceptable pepti salts

- 20 045010 dov, such as, for example, chloride or acetate (trifluoroacetate). It has been noted that the salts of the peptides in accordance with the present invention differ significantly from the peptides in their in vivo state(s), since the peptides are not salts in vivo.

The term peptide also includes the concept of oligopeptide. The term oligopeptide as used herein refers to a series of amino acid residues linked to each other, usually by peptide bonds between the alpha-amine and carbonyl groups of adjacent amino acids. The length of the oligopeptide is not particularly important to the invention as long as it retains the proper epitope or epitopes. Oligopeptides are typically less than about 30 amino acid residues in length and more than about 15 amino acids in length.

The term polypeptide refers to a series of amino acid residues, usually linked to each other by peptide bonds between the alpha-amine and carbonyl groups of adjacent amino acids. The length of the polypeptide is not particularly important to the invention as long as the proper epitopes are maintained. In contrast to the terms peptide or oligopeptide, the term polypeptide is coined to refer to molecules containing more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide encoding such a molecule is immunogenic (and thus an immunogen for the purposes of the present invention) if it is capable of inducing an immune response. In the case of the present invention, immunogenicity is more specifically defined as the ability to induce a T cell response. Thus, an immunogen will be a molecule that is capable of inducing an immune response, and, in the case of the present invention, a molecule capable of inducing a T cell response. In another aspect, the immunogen may be a peptide, a peptide-MHC complex, an oligopeptide, and/or a protein used to generate specific antibodies or TCRs against it.

The class I T cell epitope requires a short peptide that binds to the class I MHC receptor, forming a three-member complex (class I MHC alpha chain, beta-2 microglobulin and peptide) that can be recognized by a T cell carrying the appropriate T -a cellular receptor that binds to the MHC/peptide complex with suitable affinity. Peptides that bind to MHC class I molecules are typically 8-14 amino acids in length and, most typically, 9 amino acids in length.

In humans, there are three different genetic loci that encode class I MHC molecules (human MHC molecules are also called human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02 and HLA-A*07 are examples of the different MHC class I alleles that can be expressed from these loci.

Table 4

F expression frequency values for serotypes HLA-A*02, HLA-A*O1, HLA-A*03, HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44

Allele Population Calculated phenotype based on allele frequency (F) A*02 African Americans (N=28,557) 32.3% White Europeans (N=1242890) 49.3% Japanese (N=24582) 42.7% Latin Americans, South. + Center Amer. (N=146714) 46.1% Southeast Asians (N=27978) 30.4% A*01 African Americans (N=28,557) 10.2% White Europeans (N=1242890) 30.2% Japanese (N=24582) 1.8% Latin Americans, South. + Center Amer. (N=146714) 14.0% Southeast Asians (N=27978) 21.0% A*03 African Americans (N=28,557) 14.8% White Europeans (N=1242890) 26.4% Japanese (N=24582) 1.8% Latin Americans, South. + Center Amer. (N=146714) 14.4% Southeast Asians (N=27978) 10.6% A*24 African Americans (N=28,557) 2.0% White Europeans (N=1242890) 8.6% Japanese (N=24582) 35.5%

- 21 045010

Latin Americans, South. + Center Amer. (N=146714) 13.6% Southeast Asians (N=27978) 16.9% B*07 African Americans (N=28,557) 14.7% White Europeans (N=1242890) 25.0% Japanese (N=24582) 11.4% Latin Americans, South. + Center Amer. (N=146714) 12.2% Southeast Asians (N=27978) 10.4% B*08 African Americans (N=28,557) 6.0% White Europeans (N=1242890) 21.6% Japanese (N=24582) 1.0% Latin Americans, South. + Center Amer. (N=146714) 7.6% Southeast Asians (N=27978) 6.2% B*44 African Americans (N=28,557) 10.6% White Europeans (N=1242890) 26.9% Japanese (N=24582) 13.0% Latin Americans, South. + Center Amer. (N=146714) 18.2% Southeast Asians (N=27978) 13.1%

Gf haplotype frequencies were obtained from a study that used HLA typing data from a registry for more than 6.5 million US volunteer donors (Gragert et al., 2013). Haplotype frequency is the frequency of a single allele on a single chromosome. Due to the diploid set of chromosomes in mammalian cells, the frequency of occurrence of this allele in the genotype is higher and can be calculated using the Hardy-Weinberg principle (F = 1 - (1-Gf)2).

The peptides of the invention, preferably when included in a vaccine of the invention as described herein, bind to the A*02, A*01, A*03, A*24, B*07, B*08 or B*44 allele. The vaccine may also include universal peptides that bind to MHC class II. Therefore, the vaccine according to the invention can be used for the treatment of cancer in patients who are A*02-, A*01-, A*03-, A*24-, B*07-, B*08- or B*44-positive, Moreover, due to the universal binding nature of these peptides, selection of MHC class II allotypes is not necessary.

If the A*02 peptides of the invention are combined with peptides that bind to another allele, eg A*24, a higher percentage of any patient population can be treated compared to vaccination for each MHC class I allele separately. While in most populations less than 50% of patients may be covered by any one allele, a vaccine incorporating the HLA-A*24 and HLA-A*02 epitopes can treat at least 60% of patients in any relevant population. Specifically, the following percentages of patients will be positive for at least one of these alleles in various regions: USA - 61%, Western Europe - 62%, China 75%, South Korea - 77%, Japan - 86% (calculated from data www.allelefrequencies.net).

Table 5

HLA allele coverage of the Caucasian population (calculated according to (Gragert et al., 2013))

coverage (at least one Aallele) in combination with B*07 in combination with B*44 in combination with B*07 and B*44 A*02 / A*01 70% 78% 78% 84% A*02 / A*03 68% 76% 76% 83% A*02 / A*24 61% 71% 71% 80% A*'O1 / A*03 52% 64% 65% 75% A*01 / A*24 44% 58% 59% 71% A*03 / A*24 40% 55% 56% 69% A*02 / A*01 / A*03 84% 88% 88% 91% A*02 / A*01 / A*24 79% 84% 84% 89% A*02 / A*03 / A*24 77% 82% 83% 88% A*01 / A*03 / A*24 63% 72% 73% 81% A*02/A*01 /A*03/ A*24 90% 92% 93% 95%

- 22 045010

In a preferred embodiment, the term nucleotide sequence refers to a heteropolymer of deoxyribonucleotides.

The nucleotide sequence encoding a particular peptide, oligopeptide or polypeptide may be naturally occurring or may be synthesized. In general, the DNA segments encoding the peptides, polypeptides and proteins of the present invention are assembled from cDNA fragments and short oligonucleotide linkers or from a series of oligonucleotides to produce a synthetic gene that is capable of being expressed in a recombinant transcription unit including regulatory elements derived from microbial or viral operon.

As used herein, the term peptide-encoding nucleotide refers to the peptide-encoding nucleotide sequence, including artificial (man-made) start stop codons, compatible with the biological system in which the sequence is to be expressed, for example, a dendritic cell or other cellular system suitable for receiving TKR.

As used herein, reference to a nucleic acid sequence includes both single-stranded and double-stranded nucleic acid. Thus, for example, for DNA, a specific sequence, unless the context otherwise indicates, refers to single-stranded DNA of such a sequence, a duplex of such a sequence with its complement (double-stranded DNA), and the complement of such a sequence.

The term coding region refers to that region of a gene that, in natural or normal conditions, encodes the expression product of that gene in its natural genomic environment, i.e., the region that in vivo encodes the native expression product of the gene.

The coding region may be derived from an unmutated (normal), mutated or altered gene, or may even be derived from a DNA sequence, or an entire gene synthesized in the laboratory using methods well known to those skilled in the art of DNA synthesis.

The term expression product means a polypeptide or protein that is the natural product of translation of a gene and any nucleic acid sequence that encodes equivalents resulting from the degeneration of the genetic code and thus encodes the same amino acid(s).

The term fragment, when referring to a coding sequence, means a region of DNA comprising less than the entire coding region, the expression product of which retains substantially the same biological function or activity as the expression product of the entire coding region.

The term DNA segment refers to a DNA polymer, either as a single fragment or as a component of a larger DNA construct, that has been formed from DNA isolated at least once in substantially pure form, i.e., without contaminating endogenous materials and in quantity or at a concentration that allows identification, manipulation and recovery of the segment and its constituent nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such segments are provided in the form of an open reading frame, uninterrupted by internal untranslated sequences or introns that are typically present in eukaryotic genes. Untranslated DNA sequences may be present outside the open reading frame, where it does not interfere with the manipulation or expression of coding regions.

The term primer refers to a short nucleic acid sequence that can be paired with a single strand of DNA to produce a free 3'OH end at which DNA polymerase begins to synthesize the deoxyribonucleotide chain.

The term promoter refers to a region of DNA involved in binding RNA polymerase to initiate transcription.

Isolated means that the material has been removed from its original environment (eg, its natural environment if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in living organisms is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials of the natural system is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition and still be isolated such that such a vector or composition is not part of its natural environment.

The polynucleotides and recombinant or immunogenic polypeptides disclosed in accordance with the present invention may also be in purified form. The concept purified does not require absolute purity; rather, it is intended to provide a relative definition and may include highly purified preparations or only partially purified preparations, as those terms are understood by those skilled in the art. For example, individual clones isolated from a cDNA library were routinely purified to electrophoretic homogeneity. Purification of the starting material or natural material from impurities by at least one order of magnitude, preferably two or three orders of magnitude, and more preferably four or five orders of magnitude is specifically contemplated by the invention. Moreover, specifically included is a claimed polypeptide whose purity is preferably 99.999% or at least 99.99% or 99.9%; and even preferably 99% by weight or more.

Nucleic acids and polypeptides as expression products disclosed in accordance with the present invention, as well as expression vectors containing such nucleic acids and/or such polypeptides, can be in enriched form. As used herein, the term enriched means that the concentration of the material is at least about 2, 5, 10, 100 or 1000 times its natural concentration (for example), preferably 0.01%, by weight, preferably at least about 0. 1% by weight. Fortified formulations with concentrations of approximately 0.5%, 1%, 5%, 10%, and 20% by weight are also considered. The sequences, constructs, vectors, clones and other materials included in the present invention may preferably be in enriched form or isolated. The term active fragment means a fragment - typically a peptide, polypeptide or nucleic acid sequence - which produces an immune response (i.e. has immunogenic activity) when administered alone or optionally with a suitable adjuvant or in a vector to an animal such as a mammal, e.g. rabbit or mouse, also including humans; wherein such immune response takes the form of stimulation of a T cell response in a recipient animal, such as a human. Alternatively, the active fragment can also be used to initiate a T cell response in vitro.

As used herein, the terms region, segment and fragment, when used with respect to polypeptides, refer to a contiguous sequence of residues, such as amino acid residues, the sequence of which forms a subclass of a larger sequence. For example, if a polypeptide has been subjected to treatment with any of the known endopeptidases, such as trypsin or chymotrypsin, the resulting oligopeptides will represent regions, segments or fragments of the original polypeptide. When used in relation to polynucleotides, these terms refer to the products obtained by processing said polynucleotides with any of the endonucleases.

In accordance with the present invention, the concept of percent identity, or identical to percent when referring to a sequence, means that the sequence is compared to a claimed or reported sequence after aligning the compared sequence (Comparison Sequence) with the reported or reported sequence (Reference Sequence). The percentage of identity is then determined using the following formula:

percentage identity = 100 [1-(C/R)], where C is the number of differences between the Reference sequence and the Comparison sequence along the length of the alignment between the Reference sequence and the Comparison sequence, where:

(i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Comparison Sequence, and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that differs from the aligned base or amino acids in the Comparison Sequence constitute a difference; and (iii i) the alignment must start from position 1 of the aligned sequences;

and R is the number of bases or amino acids in the Reference Sequence along the length of the alignment with the Comparison Sequence with any gap occurring in the Reference Sequence also counting as a base or amino acid.

If there is a contrast between a Comparison Sequence and a Reference Sequence for which the Percentage Identity is calculated to be greater than, approximately equal to, or greater than a specified minimum Percentage Identity, then the Comparison Sequence has a specified minimum Percentage Identity with the Reference Sequence, even if alignments may exist, in of which the identity percentage calculated above is less than the established identity percentage.

As mentioned above, the present invention thus provides a peptide comprising a sequence that is selected from the group consisting of sequences having SEQ ID No. 1 to SEQ ID No. 489 or a variant thereof that is 88% homologous to the sequences of SEQ ID No. 1 to SEQ ID No. 489, or a variant thereof, which induces a cross-reaction of T cells with the specified peptide. The peptides of the invention have the ability to bind to the human major histocompatibility complex (MHC) class I molecule or - extended versions of the said peptides - to MHC class II.

In the present invention, the term homologous refers to the degree of identity (see above, Percentage Identity) between the sequences of two amino acid sequences, i.e. peptide or polypeptide sequences. The previously mentioned homology defines

- 24 045010 occurs when comparing two sequences that are compared under optimal conditions for the sequences being compared. Such sequence homology can be calculated using alignment generation, such as the ClustalW algorithm. Sequence analysis software is widely available, particularly Vector NTI, GENETYX, or other tools provided by freely available data banks.

One skilled in the art will be able to assess whether T cells induced by a variant of a particular peptide will be capable of cross-reacting with the peptide itself (Arrow et al., 2006; Colombetti et al., 2006; Fong et al., 2001; Zaremba et al. al., 1997).

By variant of a given amino acid sequence, we mean that the side chains of, for example, one or two amino acid residues are altered (for example, by replacing them with a side chain of a residue of another naturally occurring amino acid or some other side chain) such that the peptide is still capable of binding to an HLA molecule in substantially the same manner as a peptide consisting of the amino acid sequence of SEQ ID NO: 1 through SEQ ID NO: 489. For example, the peptide may be modified such that it is at least will retain, if not improve, the ability to interact with and bind to the binding groove of a suitable MHC molecule such as HLA-A*02 or -DR, and thus it will at least retain, if not improve, the ability to bind to the TCR of activated T- cells.

These T cells can then cross-react with and kill cells that express a polypeptide that contains a natural amino acid sequence of a related peptide, as defined in aspects of this invention. According to information from the scientific literature and data banks (Rammensee et al., 1999; Godkin et al., 1997), specific positions of HLA-binding peptides are typical anchor residues forming a central sequence suitable for the HLA receptor junction element, which is defined by polar, electrophysical, hydrophobic and spatial properties of the polypeptide chains forming the binding groove. Thus, one skilled in the art will be able to modify the amino acid sequences of SEQ ID No. 1 to SEQ ID No. 489, retaining known anchor residues, and will be able to determine whether such variants retain the ability to bind to MHC class I or II molecules. Variants of the present invention retain the ability to bind to the TCR of activated T cells, which can subsequently cross-react with and kill cells expressing a polypeptide that contains the natural amino acid sequence of the related peptide, as defined in aspects of the present invention.

The original (unmodified) peptides disclosed herein may be modified by substitution of one or more residues at various, possibly selected, regions along the peptide chain unless otherwise stated. Preferably, such substitutions are located at the end of the amino acid chain. Such substitutions may be conservative in nature, such as when one amino acid is replaced by an amino acid with similar structure and characteristics, as well as when a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative is the substitution of amino acids of the same or similar size and chemical character, such as the substitution of isoleucine for leucine. In studies of sequence variation within families of naturally occurring homologous proteins, certain amino acid substitutions are tolerated more often than others, and they are often associated with similarities in size, charge, polarity, and hydrophobicity between the original amino acid and its substitution; and this is the basis for defining conservative substitutions.

Conservative substitutions are defined in the context of this description as exchanges within one of the following five groups: group 1 - small, aliphatic, non-polar or weakly polar residues (Ala, Ser, Thr, Pro, Gly); group 2 - polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); group 3 - polar, positively charged residues (His, Arg, Lys); group 4 - large, aliphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); and group 5 - large, aromatic residues (Phe, Tyr, Trp).

Less conservative substitutions may involve replacing one amino acid with another that has similar characteristics but differs to some extent in size, as in the case of replacing an alanine with an isoleucine residue. Highly non-conservative substitutions may involve replacing an acidic amino acid with a polar one, or even one that is basic in nature. Such radical substitutions cannot, however, be dismissed as potentially ineffective because the chemical effects are not entirely predictable and radical substitutions may unexpectedly lead to beneficial effects not predictable based on normal chemical principles.

Of course, such substitutions may involve other structures that differ from the usual L-amino acids. Thus, D-amino acids can be replaced by L-amino acids commonly found in the antigenic peptides of the invention and also covered by the present disclosure. In addition, non-standard amino acids (ie, different from the commonly found proteinogenic amino acids) can also be used for substitution purposes to produce immunogens and immunogenic polypeptides in accordance with the present invention.

If substitutions have been made at more than one position to produce a peptide with essentially

- 25 045010 equivalent or greater antigenic activity, as defined below, then combinations of such substitutions will be analyzed to determine whether these combinations of substitutions will result in additional or synergistic effects with respect to the antigenicity of the peptide. For the most part, no more than 4 positions within a peptide should be replaced at a time.

A peptide consisting essentially of an amino acid sequence as defined herein may have one or two non-anchor amino acid substitutions (see below regarding the anchor motif) such that the ability to bind to a human major histocompatibility complex (MHC) class I or II molecule is not will be significantly altered or negatively affected compared to the unmodified peptide. In another embodiment, in a peptide consisting essentially of an amino acid sequence as defined herein, one or two amino acids may be replaced with conservative substitution partners (see information below) such that the ability to bind to a human major histocompatibility complex molecule (MHC) class I or II will not be significantly altered or negatively affected compared to the unmodified peptide.

Amino acid residues that do not significantly contribute to the interaction with the T-cell receptor can be modified by replacement with another amino acid, the inclusion of which does not significantly affect the reactivity of the T-cell and does not eliminate binding to the corresponding MHC. Thus, in addition to this condition, the peptide of the invention can be any peptide (within this term the inventors include oligopeptides or polypeptides) that includes amino acid sequences or a portion thereof or a variant thereof, as given.

Table 6

Peptide variants and motif according to SEQ ID NO: 4,

- 26 045010

- 27 045010

- 28 045010

I F M M Y M R M F V V Y V R V F T T Y T R T F Position 1 2 3 4 5 6 7 8 9 SEQ ID No. 204 A V F D TO F I R Y Option L TO L L R L F I TO I I R I F M TO M M R M F TO R F T TO T T R T F Position 1 2 3 4 5 6 7 8 9 SEQ ID No. 224 G P Q P W H A A L Option F V M A I Position 1 2 3 4 5 6 7 8 9 10

-29045010

SEQ ID No. 294 G P T P N T G A A L Option F V M A I Position 1 2 3 4 5 6 7 8 SEQ ID No 306 I L To V TO V G L Option V I M F R R V R I R M R F H H V H I H M H F R R V R I R M R F R R R R V R R I R R M R R F R H R H V R H I R H M R H F L L V L I L M L F

-30045010

- 31 045010

Longer (extended) peptides may also be suitable. It is possible that epitopes that bind to MHC class I molecules, although they are typically between 8 and 11 amino acids in length, are derived from peptide processing from longer peptides or proteins containing the true epitope. Preferably, residues that are adjacent to the true epitope do not significantly affect the proteolytic cleavage required for presentation of the true epitope during processing.

The peptides of the invention can be extended with up to four amino acids, which means that 1, 2, 3 or 4 amino acids can be added to one of the ends in any combination between 4:0 and 0:4. Combinations of elongations in accordance with the invention can be taken from table. 7.

Table 7 ________Peptide elongation combinations according to the invention________

C-end N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4 N-terminus C-end 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for elongation/extension can be peptides from the original protein sequence or any other amino acid(s). Elongation can be used to increase the stability or solubility of peptides.

Thus, the epitopes of the present invention may be identical to naturally occurring tumor-associated or tumor-specific epitopes, or may include epitopes that differ by no more than four residues from the control peptide, provided that they have substantially identical antigenic activity.

- 32 045010

In an alternative embodiment, the peptide is extended on one side or the other, or on both sides simultaneously, by adding more than 4 amino acids, preferably to a total length of up to 30 amino acids. This can lead to the formation of peptides that bind to MHC class II. Binding to MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and epitopes of peptide variants that bind to MHC class I molecules, where the peptide or variant has a total length of 8 and 100, 9 and 100, 10 and 100, 11 and 100, 12 and 100 , preferably from 8 and 30 and from 9 and 30, from 10 and 30, from 11 and 30, from 12 and 30, most preferably from 8 and 14, from 9 and 14, from 10 and 14, from 11 and 14 , from 12 and 14. The present invention further provides peptides and peptide variant epitopes that bind to MHC class I molecules, wherein the peptide or variant has a total length of specifically 8, 9, 10, 11, 12, 13, or 14 amino acids, in In the case of extended peptides that bind to MHC class II molecules, the length may also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant in accordance with the present invention will have the ability to bind to a human major histocompatibility complex (MHC) class I or II molecule. The binding of the peptide or variant to the MHC complex can be tested by methods known in the art.

Preferably, T cells specific for the peptide of the present invention are tested against substituted peptides; the peptide concentration at which the substituted peptides achieve half the maximum lysis growth relative to background is no more than about 1 mM, preferably no more than about 1 μM, more preferably no more than about 1 nM, and even more preferably no more than about 1 nM 100 pM and, most preferably, no more than about 10 pM. It is also preferred that the substituted peptide is recognized by T cells of more than one individual, at least two and more preferably three individuals.

In a particularly preferred embodiment of the invention, the peptide consists or essentially consists of an amino acid sequence in accordance with SEQ ID No. 1 to SEQ ID No. 489.

Consisting essentially of means that the peptide in accordance with the present invention, in addition to any of the sequences of SEQ ID No. 1 to SEQ ID No. 489, or a variant thereof, contains additional N- and/or C-terminal amino acid sequence fragments that do not necessarily form part of the peptide that functions as an epitope for MHC molecules.

However, these fragments may be important to ensure efficient administration of the peptide of the present invention into cells. In one embodiment of the present invention, the peptide is part of a fusion protein that includes, for example, the N-terminal 80 amino acids of the antigen-associated invariant chain (a33, hereinafter li) of HLA-DR, as taken from the NCBI data bank, accession number - GenBank Accession -number X00497. In other types of fusion, the peptides of the present invention may be fused to an antibody described herein, or a functional portion thereof, specifically inserted into the sequence of an antibody so as to be a specific target of said antibody, or, for example, fused to or incorporated into an antibody which is specific for dendritic cells described in this application.

In addition, the peptide or variant may be further modified to improve stability and/or binding to MHC molecules to produce a stronger immune response. Methods for such peptide sequence optimization are well known in the art and include, for example, the introduction of reversed peptide or non-peptide bonds.

In a reversed peptide bond, the amino acid residues are joined by non-peptide bonds (-CO-NH-), but the peptide bond is reversed. Such retro-reverse peptidomimetics can be prepared by methods known in the art, such as those described by Meziere et al. (1997) (Meziere et al., 1997), incorporated herein by reference. This approach covers the production of pseudopeptides that contain changes involving the backbone but not the orientation of the side chains. Meziere et al. (Meziere et al., 1997) show that these pseudopeptides are useful for binding to MHC and inducing T helper cell responses. Retro-reverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.

Non-peptide bonds are, for example, -CH ₂ -NH, -CH2S-, -CH2CH2-, -CH=CH-, -COCH2-, -CH(OH)CH ₂ - and -CH2SO-. US Patent No. 4,897,445 proposes a method for the solid-phase synthesis of non-peptide bonds (-CH ₂ -NH) in polypeptide chains, which includes polypeptides synthesized using standard techniques and a non-peptide bond synthesized by the reaction of an aminoaldehyde and an amino acid in the presence of NaCNBH ₃ .

Peptides comprising the sequences described above can be synthesized with additional chemical groups at their amine and/or carboxyl termini to increase the stability, bioavailability and/or affinity of the peptides. For example, hydrophobic groups such as carbobenzoxyl, dansyl or tert-butyloxycarbonyl groups can be added to the amine termini of the peptides. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group can be placed at the amine termini of peptides. Except

- 33 045010 In addition, a hydrophobic group, tert-butyloxycarbonyl or amide group can be added to the carboxyl ends of the peptides.

In addition, all peptides according to the invention can be synthesized in order to change their spatial configuration. For example, the D-isomer of one or more amino acid residues of the peptide may be used rather than the usual L-isomer. Moreover, at least one of the amino acid residues of the peptides of the invention may be replaced by one of the well-known non-naturally occurring amino acid residues. Changes, such as these, may serve to improve the stability, bioavailability and/or binding properties of the peptides of the invention.

Likewise, a peptide or variant of the invention may be chemically modified by reacting individual amino acids either before or after synthesis of the peptide. Examples of such modifications are well known in the art and are summarized, for example, in R. Lundblad, Chemical Reagents for Protein Modification, 3rd ed. KPK Press, 2004 (Lundblad, 2004), which is included in the description by reference. Chemical modification of amino acids includes, but is not limited to, modification by acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amine groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amide modification of carboxyl groups, and sulfhydryl modification by oxidation of cysteine with performic acid to cysteic acid, formation of mercury derivatives, formation of mixed disulfides with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide, and carbamoylation with cyanate at alkaline pH, although not limited to them. In this regard, one skilled in the art may consult Chapter 15 in Current Protocols In Protein Science, Eds. Hassan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995) for more information on techniques associated with the chemical modification of proteins.

Briefly, modification of, for example, arginyl residues in proteins is often based on the reaction of vicinal dicarbonyl compounds such as phenylglyoxal, 2,3-butanedione and 1,2-cyclohexanedione to form an adduct. Another example is the reaction of methylglyoxal with arginine residues. Cysteine can be modified without concomitant modification of other nucleophilic sites such as lysine and histidine. As a result, a large number of reagents are available for cysteine modification. Websites of companies such as Sigma-Aldrich (http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during thermal processing of biopharmaceuticals. Woodward's K-reagent can be used to modify specific glutamic acid residues.

N-(3-(Dimethylaminopropyl)-N'-ethylcarbodiimide can be used to form intramolecular cross-links between a lysine residue and a glutamic acid residue. For example, diethyl pyrocarbonate is a reagent for modifying histidyl residues in proteins. Histidine can also be modified using 4-hydroxy -2-nonenal. The reaction of lysine residues and other α-amine groups is useful, for example, in the binding of peptides to surfaces or the cross-linking of proteins/peptides. Lysine is the attachment site for polyethylene glycol and the main modification site in the glycosylation of proteins. Methionine residues in proteins can be modified , for example, with iodoacetamide, bromoethylamine and chloramine T.

Tetranitromethane and N-acetylimidazole can be used to modify tyrosyl residues. Cross-linking through the formation of dityrosine can be achieved using hydrogen peroxide/copper ions.

Recent tryptophan modification studies have used N-bromosuccinimide, 2hydroxy-5-nitrobenzyl bromide, or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNSskatole).

Successful modification of therapeutic proteins and peptides with PEG (polyethylene glycol) is often associated with increased circulation half-life, while cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde is used to produce hydrogels. Chemical modification of allergens for immunotherapy is often achieved by carbamoylation with potassium cyanate.

A peptide, or a variant in which the peptide is modified or includes non-peptide linkages, is a preferred embodiment of the invention.

Another embodiment of the present invention relates to a non-naturally occurring peptide, wherein said peptide consists of or consists essentially of the amino acid sequence of SEQ ID No: 1 to SEQ ID NO: 489 and has been produced synthetically (e.g., synthesized) into a pharmaceutically acceptable form salt. Methods for synthetically producing peptides are well known in the art. The peptide salts of the present invention differ significantly from peptides in their in vivo state(s) since the synthesized peptides are not

- 34 045010 salts in vivo. The non-naturally occurring salt form of the peptide mediates the solubility of the peptide, particularly in the context of pharmaceutical compositions comprising peptides, such as the peptide vaccines disclosed herein. Sufficient and at least significant solubility of the peptide(s) is necessary for effective administration of the peptides to the subject to be treated. Preferably, the salts are pharmaceutically acceptable peptide salts. Salts in accordance with the invention include alkali ^and alkaline earth salts, such as salts of the Hofmeister series, including as anions PO ₄ ^3- , SO4 ^2- , ^CH3COO- , Cl- ^, Br- ^{, NO3-} , ^ClO4- , ^I- , SCN- and as cations NH4 ⁺ , Rb ⁺ , K ⁺ , Na ⁺ , Cs ⁺ , Li ⁺ , Zn ²⁺ , Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Cu ²⁺ and Ba ²⁺ . In particular, the salts are selected from (NH4)3PO4, (NH4)2HPO4, (NH4)H2PO4, (NH4)2SO4, NH4CH3COO, NHCl, NH4Br, NH4NO3, NH4QO4, NH4I, NH4SCN, Rb3PO4, Rb2HPO4, RbH2PO4, Rb2SO4, Rb4CH3COO, RB4CL, RB4BR, RB4NO3, RB4CLO4, RB4I, RB4SCN, K3PO4, K2HPO4, KH2PO4, K2SO4, KCH3COO, KBR, KNO3, KCLO4, KI, KSCN, Na3PO4, Na2HPO4, NaH2PO4, NAH2PO4, NAH2PO4, NAH2PO4, NAH2PO4, NAH2PO4, NAH2PO4, NAH Na2SO4, Nach3COO, NACL, Nabr, Nano3, NaClO4, NaI, NaSCN, ZnCl2 Cs3PO4, Cs2HPO4, CsH2PO4, Cs2SO4, CsCH3COO, CsCl, CsBr, CsNO3, CsClO4, CsI, CsSCN, Li3PO4, Li2HPO4, LiH2PO4, Li2SO4, LiCH3COO, LiCl, LiBr, LiNO3, LiClO4, LiI ,LiSCN , Cu2SO4, Mg3(PO4)2, Mg2HPO4, Mg(H2PO4)2, Mg2SO4, Mg(CH3COO)2, MgCl2, MgBr2, Mg(NO3)2, Mg(ClO4)2, MgI2, Mg(SCN)2, MnCl2 , Ca3(PO4), Ca2HPO4, Ca(H2PO4)2, CaSO4, Ca(CH3COO)2, CaCl2, CaBr2, Ca(NO3)2, Ca(ClO4)2, CaI2, Ca(SCN)2, Ba3(PO4) 2, Ba2HPO4, Ba(H2PO4)2, BaSO4, Ba(CH3COO)2, BaCl2, BaBr2, Ba(NO3)2, Ba(ClO4)2, BaI2, and Ba(SCN) ₂ . Particularly preferred are NH acetate, MgCl ₂ , KH ₂ PO ₄ , Na ₂ SO ₄ , KCl, NaCl and CaCl ₂ , such as for example the chloride or acetate (trifluoroacetate) salts.

In general, peptides and variants (at least those containing peptide bonds between amino acid residues) can be synthesized by the Fmoc-polyamide solid-phase peptide synthesis method as disclosed by Lukas et al. (Lukas et al., 1981) and in the accompanying references. Temporary protection of the N-amino group is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Re-cleavage of this highly alkali-labile protecting group is achieved using 20% piperidine in N,N-dimethylformamide. Side chain functional groups can be protected to form compounds such as their butyl esters (in the case of serine, threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and a derivative of 4-methoxy-2,3,6-trimethylbenzenesulfonyl (in the case of arginine). When glutamine or asparagine are C-terminal residues, a 4,4'dimethoxybenzhydryl group is used to protect the side chain amido group. The solid-phase carrier is based on the polymer polydimethylacrylamide, consisting of three monomers: dimethylacrylamide (framework monomer), bisacryloylethylenediamine (cross-linking component, linker) and acryloyl sarcosine methyl ester (functionalizing agent). To form an easily cleavable bond between the peptide and the resin, an acid-labile derivative of 4-hydroxymethylphenoxyacetic acid is used. All amino acid derivatives are added as presynthesized symmetric anhydride derivatives with the exception of asparagine and glutamine, which are added using an N,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole-mediated reverse coupling reaction. All coupling and deprotection reactions were monitored using ninhydrin, trinitrobenzenesulfonic acid or isotene control methods. Once synthesis is complete, the peptides are cleaved from the carrier resin with concomitant side chain deprotection by treatment with 95% trifluoroacetic acid containing a 50% scavenger mixture. Commonly used scavengers include ethanedhiol, phenol, anisole and water, the final choice depending on the amino acid constituents of the peptide being synthesized. A combination of solid-phase and liquid-phase methods for peptide synthesis is also possible (see, for example, (Bruckdorfer et al., 2004), and the accompanying references).

Trifluoroacetic acid is removed by evaporation in vacuo followed by grinding with diethyl ether to obtain the crude peptide. Any scavengers present are removed by a simple extraction technique, which allows the crude peptide without scavengers to be obtained after lyophilization of the aqueous phase. Peptide synthesis reagents are generally available from, for example, Calbiochem-Novabiochem (Nottingham, UK).

Purification can be accomplished by any technique or combination of techniques such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and (usually) reverse phase high performance liquid chromatography using, for example, acetonitrile/water gradient separation.

Peptide analysis can be carried out using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (SPE), reverse phase high performance liquid chromatography, amino acid analysis after acid hydrolysis and fast atom bombardment (FAB) mass spectrometry analysis, as well as MALDI mass spectrometry analysis and ESI-Q-TOF.

To select over-presented peptides, a presentation profile was calculated to estimate the median sample presentation as well as the variation across repeated measurements. The profile compares samples of the tumor form of interest with background levels of

- 35 045010 samples of normal tissue. Each of these profiles can then be consolidated into an overpresentation score by calculating a p value from a linear mixed-effects model (Pinheiro et al., 2015), adjusting for repeated analyzes for the false-positive rate (Benjamini and Hochberg, 1995) (cf. example 1, Fig. 1).

To identify and relative quantify HLA ligands by mass spectrometry analysis, HLA molecules from shock-frozen tissue samples were purified and HLA-associated peptides were isolated from them. The isolated peptides were separated and sequences were identified using real-time liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (nanoESI) techniques. The obtained peptide sequences were confirmed by comparing the fragmentation patterns of natural tumor-associated TUMAP peptides recorded on samples (N = 201 samples) of lung cancer (including NSCLC and SCLC) with the fragmentation patterns of corresponding synthetic control peptides with identical sequences. Because the peptides were identified directly as ligands of HLA molecules from primary tumors, these results provide direct evidence of natural processing and presentation of the identified peptides on primary cancer tissue obtained from 201 lung cancer patients (including NSCLC and SCLC).

The XPRESIDENT® v2.1 drug pipeline technology platform (see, for example, US Patent Application 2013-0096016, incorporated herein by reference in its entirety) allows for the identification and selection of appropriate over-presented peptides as drug candidates. vaccines based on direct relative quantification of HLA-restricted peptide levels on cancer tissue compared to several different non-cancerous tissues and organs. This was accomplished by developing differential quantitation based on label-free LC-MS data processed by a proprietary data analysis technology platform integrating algorithms for sequence identification, spectral clustering, ion counting, retention time alignment, and deconvolution. states of charge and normalization.

Presentation levels, including bias estimates, were calculated for each peptide and sample. Peptides that are presented exclusively on tumor tissue and peptides that are excessively presented on tumor tissues compared to non-cancer tissues and organs were identified.

HLA-peptide complexes from lung cancer tumor tissue samples (including NSCLC and SCLC) were purified; HLA-associated peptides were isolated and analyzed by LC-MS (see Example 1). All TUMAPs contained in this patent application were identified using this approach in lung cancer samples (including NSCLC and SCLC), confirming their presentation on lung cancer cells (including NSCLC and SCLC).

TUMAP peptides identified in numerous lung cancer (including NSCLC and SCLC) and normal tissue samples were quantitatively analyzed by isotopically label-free LC/MS using ion counting. The method is based on the assumption that the peak areas of a peptide when analyzed by LC/MS are correlated with its abundance in the sample. All quantitative peptide signals from different LC/MS experiments were normalized based on the main trend, their average value per sample was calculated, and summarized in a histogram in the so-called. presentation profile. The presentation profile consolidates various analysis methods such as protein data bank searching, spectral clustering, charge state (discharge) deconvolution and retention time equalization and normalization.

In addition to overpresentation of the peptide, the expression of the parent gene mRNA was examined. mRNA data obtained by RNA sequencing (RNASeq) from normal tissues and cancer tissues (cf. example 2, Fig. 2). An additional source of normal tissue data was a publicly available RNA expression database of approximately 3000 normal tissue samples (Lonsdale, 2013). Peptides that are derived from proteins that are encoded by mRNA that exhibits high levels of expression in cancer tissue but very low levels or absence in vital normal tissues have been included as preferred in the present invention.

The present invention provides peptides that are useful for the treatment of cancers/tumors, preferably lung cancer (including NSCLC and SCLC), the cells of which present in excess or exclusively the peptides of the invention. As shown by mass spectrometric analysis, these peptides were naturally presented by HLA molecules on samples of primary human lung cancer (including NSCLC and SCLC).

Many of the parent genes/proteins (also called full-length proteins or core proteins) from which the peptides were derived were highly overexpressed in cancer cells compared to normal tissues - the term normal tissues in connection with the present invention means healthy lung cells or other normal tissue cells demonstrating a high degree of association of parent genes with the tumor (see example 2). Moreover, the peptides themselves are highly over-presented on tumor tissue - the concept of tumor tissue in

- 36 045010 in connection with the present invention involves a tissue sample from a patient suffering from lung cancer (including NSCLC and SCLC), but not on normal tissue (see example 1).

HLA-associated peptides can be recognized by the immune system, specifically by T lymphocytes. T cells can destroy cells presenting a recognized HLA/peptide complex; for example, lung cancer cells (including NSCLC and SCLC), presenting the resulting peptides.

The peptides of the present invention have been shown to be capable of stimulating T cell responses and/or are over-presented and can therefore be used to produce antibodies and/or TCRs, such as soluble TCRs, in accordance with the present invention (see Example 3, Example 4). In addition, the peptides, when complexed with an appropriate MHC molecule, can be used to produce antibodies and/or TCRs, in particular soluble TCRs, in accordance with the present invention. Appropriate methods are well known to those skilled in the art and can also be found in the relevant literature (see also below). Thus, the peptides of the present invention are useful for generating an immune response in a patient to destroy tumor cells. An immune response in a patient can be induced by direct administration of the described peptides or suitable precursor substances (eg, extended peptides, proteins or nucleic acids encoding these peptides) to the patient, ideally in combination with an immunogenicity enhancing substance (i.e. adjuvant). It can be expected that the immune response induced by such therapeutic vaccination will be highly specifically directed against tumor cells, since the target peptides of the present invention are not presented on normal tissues in comparable copy numbers, thereby preventing the risk of unwanted autoimmune reactions against normal cells in patient.

The present description further relates to T cell receptors (TCRs) including an alpha chain and a beta chain (alpha/beta TCR). Also provided are peptides according to the invention that are capable of binding to TCRs and antibodies if they are presented by an MHC molecule.

The present description also relates to TCR fragments of the invention that are capable of binding to a peptide antigen of the invention when presented by an HLA molecule. This term particularly refers to soluble TCR fragments, for example, TCRs without transmembrane segments and/or constant regions, single-chain TCRs and their fusion products, for example, with Ig.

The present description also relates to the TCR expression nucleic acids, vectors and host cells and peptides of the present invention; and methods of their application.

The term T cell receptor (abbreviated as TCR) refers to a heterodimeric molecule comprising an alpha polypeptide chain (alpha chain) and a beta polypeptide chain (beta chain), where the heterodimeric receptor is capable of binding to a peptide antigen presented by an HLA molecule. This concept also includes the so-called gamma/delta TCRs.

In one embodiment <...> there is provided a method for producing a TCR as described herein, the method comprising culturing a host cell capable of expressing the TCR under conditions suitable for stimulating TCR expression.

The present description, in another aspect, further relates to methods according to the present description, wherein the antigen is loaded on MHC class I or II molecules expressed on the surface of a suitable antigen presenting cell or artificial antigen presenting cell, when a sufficient amount of the antigen is contacted with the antigen presenting cell, or the antigen is loaded to MHC class I or II tetramers by tetramerization of antigen-monomer complexes of MHC class I or II.

The alpha and beta chains of the alpha/beta TCR and the gamma and delta chains of the gamma/delta TCR are generally considered to have two domains each, namely variable and constant domains. The variable domain consists of a sequential variable segment (V) and a connecting segment (J). The variable domain may also include a leader segment (L). Beta and delta chains may also include diversity segments (D). The alpha and beta constant domains may also include C-terminal transmembrane (TM) domains that anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term TCR gamma variable domain, as used in the context of this invention, refers to the junction of the TCR gamma V (TRGV) segment without leader segment (L) and the TCR gamma J (TRGJ) segment, and the term constant domain TCR gamma refers to the extracellular segment of TRGC or the C-terminal truncated sequence of TRGC. Equally, the term TCR delta variable domain refers to the junction of the TCR delta V (TRDV) segment without leader segment (L) and the TCR delta D/J segment (TRDD/TRDJ), and the term TCR delta constant domain refers to the extracellular segment TRDC or C-terminal truncated sequence.

The TCRs of the present invention preferably bind to the peptide-HLA molecule complex with a binding affinity (KD) of about 100 μM or lower, about 50 μM or lower, about 25 μM or lower, or about 10 μM or lower. High-affinity TCRs with af are more preferable

- 37 045010 binding affinity of about 1 μM or lower, about 100 nM or lower, about 50 nM or lower, about 25 nM or lower. Non-limiting examples of preferred binding affinity ranges for the TCRs of the present invention include from about 1 nM to about 10 nM; from about 10 nM to about 20 nM; from about 20 nM to about 30 nM; from about 30 nM to about 40 nM; from about 40 nM to about 50 nM; from about 50 nM to about 60 nM; from about 60 nM to about 70 nM; from about 70 nM to about 80 nM; from about 80 nM to about 90 nM; and from about 90 nM to about 100 nM.

The term specific binding, as used in connection with the concept of a TCR of the present invention, and its grammatical variants are used to refer to a TCR with a binding affinity (KD) for a peptide-HLA molecule complex of 100 μM or lower.

Alpha/beta heterodimeric TCRs according to the present description may have an introduced disulfide bond between their constant domains. Preferred TCRs of this kind include those having a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, unless Thr 48 of the TRAC domain and Ser 57 of the TRBC1 or TRBC2 domains are replaced by cysteine residues, wherein said cysteine residues form a disulfide bond between the sequence TRAC constant domain and the TRBC1 or TRBC2 TCR constant domain sequence.

With or without the introduction of the interstrand linkage mentioned above, the alpha/beta heterodimeric TCRs of the present invention may have a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, and a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence The TCR may be linked by the occurring in nature, a disulfide bond between Cys4 of exon 2 of the TRAC domain and Cys2 of exon 2 of the TRBC1 or TRBC2 domain.

The TCRs of the present invention may include a detectable label selected from the group consisting of a radionuclide, a fluorophore, and a biotin. The TCRs of the present invention may be conjugated to a therapeutically active ingredient, such as a radionuclide, chemotherapeutic agent or toxin.

In one embodiment, a TCR of the present invention having at least one alpha chain mutation and/or having at least one beta chain mutation has modified glycosylation compared to a TCR without the mutations.

In one embodiment, a TCR containing at least one mutation in the TCR alpha chain and/or the TCR beta chain has a binding affinity for and/or a binding half-life for the peptide-HLA molecule complex that is at least twice as high higher than that of TCR containing TCR alpha chain without mutations and/or TCR beta chain without mutations. Enhancing the affinity of tumor-specific TCRs, as well as its use, relies on the existence of a window with optimal affinity parameters for TCRs. The existence of such a window is based on the observation that TCRs specific for HLA-A2-restricted pathogens have KD rates that are generally approximately 10-fold lower compared to TCRs specific for HLA-A2-restricted tumor-associated autoantigens. For pathogen-specific TCRs restricted by other alleles compared to tumor-associated autoantigens, the KD value may be in a slightly different range, but there are no fundamental differences in the possibility of creating TCRs between different alleles. It is now known that although tumor antigens have immunogenic potential because tumors arise from an individual's own cells, only mutant proteins or proteins with changes in translational processing will be perceived as foreign by the immune system. Antigens that are elevated or expressed in excess (so-called autoantigens) will not necessarily elicit a functional immune response against the tumor: T cells expressing TCRs that are highly active against these antigens will be subject to internal negative selection thymus gland in a process known as central tolerance, which means that only T cells with low affinity TCRs for self-antigens will remain. Therefore, the affinity of TCRs or variants according to the present description for peptides can be enhanced by methods well known in the art.

The present description further relates to a method for identifying and isolating TCRs in accordance with the present description, the method comprising incubating PBMCs from healthy donors negative for the present allele with monomeric HLA/peptide complexes, incubating PBMCs with tetramer-phycoerythrin (PE) and isolating High avidity T cells using fluorescence activated cell sorting (FACS)-Calibur.

The present description further relates to a method for identifying and isolating TCRs in accordance with the present description, the method comprising producing a transgenic mouse with entire human TCRae gene loci (1.1 and 0.7 million bp), the T cells of which express various human TCRs, compensating for mouse TCR deficiency, immunizing the mouse with the peptide, incubating transgenic mouse-derived PBMCs with tetramer-phycoerythrin (PE), and isolating high-avidity T cells using fluorescence-activated cell sorting (FACS)-Calibur.

In one aspect, to obtain T cells expressing TCRs as described herein,

- 38 045010 nucleic acids encoding TCR-alpha and/or TCR-beta chains according to the present description are cloned into expression vectors such as gamma-retrovirus or -lentivirus. Recombinant viruses are produced and tested for their functionality, such as antigen specificity and functional avidity. An aliquot of the final product is then used to transduce a target population of T cells (usually purified from the patient's PBMCs), which are cultured before infusion into the patient.

In another aspect, to obtain T cells expressing TCRs as described herein, TCR RNA is synthesized using techniques known in the art, such as in vitro transcription systems. In vitro synthesized TCR RNAs are then introduced by electroporation into primary CD8+ T cells obtained from healthy donors to re-express the alpha and/or beta chains of tumor-specific TCRs.

To increase the level of expression, nucleic acids encoding TCRs according to the present description can be operably linked to strong promoters, such as long terminal repeats of retrovirus (LTR), cytomegalovirus (CMV), mouse stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK ), β-actin, ubiquitin and the combined simian virus 40 (SV40)/CD43 promoter, elongation factor (EF)-1a and spleen necrosis virus (SFFV) promoter. In a preferred embodiment, the promoter is heterologous to the nucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes as described herein may contain additional elements that can enhance transgene expression, including the central polypurine tract (cPPT), which promotes nuclear translocation of lentiviral constructs (Follenzi et al., 2000), and post-transcriptional regulatory woodchuck hepatitis virus element (wPRE), which increases the level of transgene expression by increasing RNA stability (Zufferey et al., 1999) (Zufferey et al., 1999).

The alpha and beta chains of the TCRs of the present invention may be encoded by nucleic acids located in separate vectors, or may be encoded by polynucleotides located in the same vector.

Achieving high levels of TCR surface expression requires transcription of high levels of both TCR-alpha and TCR-beta chains introduced by the TCR. To achieve this, the TCR-alpha and TCR-beta chains of the present disclosure can be cloned into bicistronic constructs in a single vector that has been shown to overcome this obstacle. The use of the viral internal ribosome entry site (IRES) between the TCR-alpha and TCR-beta chains results in coordinated expression of both chains, since the TCR-alpha and TCR-beta chains are generated from a single transcript, which is split into two proteins during transcription, producing equal molar ratio of TCR-alpha and TCR-beta chains (Schmitt et al., 2009).

Nucleic acids encoding TCRs as described herein can be codon-optimized to increase expression by the host cell. Redundancy in the genetic code allows some amino acids to be encoded by more than one codon, but some specific codons are less optimal than others due to the relative availability of suitable tRNAs as well as other factors (Gustafsson et al., 2004). Modification of the TKR-alpha and TKR-beta gene sequences so that each amino acid is encoded by the optimal codon for mammalian gene expression, as well as the removal of unstable mRNA motifs or cryptic splice sites, have been shown to significantly enhance the expression of TKR-alpha and TKR-beta genes ( Scholten et al., 2006).

In addition, disruption of complementarity between introduced and endogenous TCR chains can lead to the acquisition of specificities that pose a significant risk for autoimmunity. For example, the formation of mixed TCR dimers can reduce the number of CD3 molecules available to form correctly paired TCR complexes and thus can significantly reduce the functional avidity of cells expressing the introduced TCR (Kuball et al., 2007).

To reduce mispairing, the C-terminal domain of the introduced TCR chains according to the present disclosure can be modified to promote interchain affinity while reducing the ability of the introduced chains to pair with endogenous TCR. These strategies may include replacing the C-terminal domains of the human TCR-alpha and TCR-beta chains with their murine equivalents (C-terminal murinized domain); obtaining a second interchain disulfide bond in the C-terminal domain by introducing a second cysteine residue into both chains: TCR-alpha and TCR-beta of the introduced TCR (cysteine modification); exchange of interacting residues in the C-terminal domain of TCRalpha and TCR-beta chains (knob-to-cavity); and fusion of the variable domains of TCR-alpha and TCR-beta chains directly into CD3Z (CD3Z fusion) (Schmitt et al., 2009).

In one embodiment, the host cell is genetically modified to express a TCR as described herein. In preferred embodiments, the host cell

- 39 045010 is a human T cell or T cell precursor. In some embodiments, the T cell or T cell precursor is obtained from a patient with cancer. In other embodiments, the T cell or T cell precursor is obtained from a healthy donor. The host cells herein may be allogeneic or autologous to the patient being treated. In one embodiment, the host cell is a gamma/delta T cell transformed to express an alpha/beta TCR.

The pharmaceutical composition is a composition suitable for administration to a human in a healthcare setting. Preferably, the pharmaceutical composition is sterile and manufactured in accordance with GMP (Good Manufacturing Practice) regulations.

Pharmaceutical compositions include peptides both in free form and in pharmaceutically acceptable salt form (see also above). In one aspect, the peptide described herein is in the form of a pharmaceutically acceptable salt. In another aspect, the peptide is provided in the form of a pharmaceutical salt in crystalline form.

In one aspect, a pharmaceutically acceptable salt as described herein refers to salts having toxicity profiles in the range that are acceptable for pharmaceutical use.

As used herein, the term pharmaceutically acceptable salt refers to derivatives of the disclosed peptides, wherein the peptide is modified by producing acidic or basic salts of the substance. For example, acid salts are prepared from the free base (typically where the neutral form of the drug has a neutral -NH2 group) using reaction with a suitable acid. Suitable acids for the preparation of acid salts include organic acids, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, ptoluenesulfonic acid, salicylic acid and the like, and inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Conversely, the preparation of base salts of acid components that may be present on the peptide is accomplished using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine and the like.

In one aspect, pharmaceutically acceptable salts can enhance the solubility and/or stability of the peptides described herein. In another aspect, the pharmaceutical salts described herein can be prepared by conventional means from an appropriate carrier peptide or complex by reacting, for example, a suitable acid or base with the peptides or complexes described herein. In another aspect, the pharmaceutically acceptable salts are provided in crystalline or semicrystalline form. In yet another aspect, pharmaceutically acceptable salts may include, for example, those described in the Handbook of Pharmaceutical Salts: Properties, Selection, and Use by P. H. Stahl and C. G. Wermuth (Wiley-VCH 2002) and L. D. Bighley, S. M. Berge, D. S. Monkhouse, in Encyclopedia of Pharmaceutical Technology. Eds. J. Swarbrick and J. S. Boylan, Vol. 13, Marcel Dekker, Inc., New York, Basel, Hong Kong 1995, pp. 453-499, each of these references is incorporated herein by reference in its entirety.

In one particularly preferred embodiment, the pharmaceutical compositions include peptides in the form of salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is an immunotherapeutic drug such as a vaccine. It can be administered directly to the patient, into the affected organ, or systemically via the intravenous, intramuscular, subcutaneous, intravenous, and intravenous routes, or ex vivo into patient-derived cells or a human cell line, which can then be administered to a patient or used in vitro to select a subpopulation from patient-derived immune cells, which are then reintroduced to the patient. If the nucleic acid is introduced into cells in vitro, it may be advantageous for the cells to be transfected to co-express immunostimulatory cytokines such as interleukin-2. The peptide may be substantially pure or in combination with an immunostimulatory adjuvant (see below) or used in combination with immunostimulatory cytokines or administered with a suitable delivery system, such as liposomes. The peptide may also be conjugated to a suitable carrier such as fissurella hemocyanin (KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). The peptide may also be labeled or may be a fusion protein or hybrid molecule. Peptides whose sequence is given in the present invention are expected to stimulate CD4+ or CD8+ T cells. However, CD8 T cell stimulation is more effective in the presence of support provided by CD4 helper T cells. Thus, for MHC class I epitopes that stimulate CD8 T cells, fusion partners or regions of the fusion molecule appropriately provide epitopes that stimulate CD4

- 40 045010 positive T cells. CD4 and CD8 stimulating epitopes are well known in the art and include those identified in the present invention.

In one aspect, the vaccine includes at least one peptide having the amino acid sequence of SEQ ID No. 1 of SEQ ID No. 489, and at least one additional peptide, preferably two to 50, more preferably two to 25, even more preferably two to 20, and most preferably two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or eighteen peptides. The peptide(s) may be derived from one or more specific TAAs and may bind to MHC class I molecules.

In yet another aspect of the invention, there is provided a nucleic acid (eg, a polynucleotide) encoding a peptide or variant of a peptide of the invention. The polynucleotide can be, for example, DNA, cDNA, PNA, RNA or combinations thereof, both single- and/or double-stranded; natural or stabilized forms of polynucleotides, such as, for example, polynucleotides with a phosphorothioate backbone, and may or may not contain introns, provided that the polynucleotide encodes a peptide. Of course, only peptides that contain naturally occurring amino acid residues linked by naturally occurring peptide bonds can be encoded by a polynucleotide. In another aspect of the invention, an expression vector capable of expressing a polypeptide in accordance with the invention is provided.

A number of methods have been developed for linking polynucleotides, particularly DNA, to vectors, for example using complementary overhangs. For example, complementary homopolymer tails can be added to a DNA segment for insertion into vector DNA. This vector and the DNA segment are then connected by a hydrogen bond between complementary homopolymer tails, forming recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide an alternative method for joining a DNA segment to vectors. Synthetic linkers containing a number of restriction endonuclease recognition sites are commercially available from various sources, including International Biotechnologies Inc, New Haven, CT, USA.

A desired method for modifying DNA encoding a polypeptide of the invention uses polymerase chain reaction as disclosed in Saiki RK et al. (Saiki et al., 1988). This method can be used to introduce DNA into a suitable vector, for example by designing at suitable restriction sites, or it can be used to modify DNA in other suitable ways known in the art. If viral vectors are used, poxviral or adenoviral vectors are preferred.

The DNA (or in the case of retroviral RNA vectors) can then be expressed in a suitable host to produce a polypeptide comprising the peptide or variant of the invention. Thus, DNA encoding a peptide or variant of the invention can be used in accordance with known techniques, modified as appropriate in light of the teachings disclosed herein, to construct an expression vector, which is then used to transform a suitable host cell for expression and production polypeptide according to the invention. Such techniques include those disclosed, for example, in U.S. Patent Nos. 4,440,859, 4,530,901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide of a compound of the invention can be linked to a wide variety of other DNA sequences for introduction into the appropriate host. The DNA companion will depend on the nature of the host, the method of introducing the DNA into the host, and whether maintenance in episomal or integrative form is desired.

Typically, the DNA is introduced into an expression vector, such as a plasmid, with the appropriate orientation and correct reading frame for expression. If desired, the DNA may be linked to appropriate transcriptional and translational coordination nucleotide sequences recognized by the desired host, although such control elements are usually present in the expression vector. The vector is then introduced into the host using standard methods. As a rule, not all hosts are transformed by the vector. It will therefore be necessary to isolate the transformed host cells. One selection technique involves introducing into an expression vector a DNA sequence, with any necessary control elements, that encodes a selected trait in the transformed cell, such as antibiotic resistance.

Alternatively, the gene for such a selectable trait may be on another vector that is used to co-transform the desired host cell.

Host cells that have been transformed with the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art, taking into account the teachings disclosed herein, leading to expression of the polypeptide, which can then be isolated.

Many expression systems are known, including bacteria (for example, E. coli and Bacillus

- 41 045010 subtilis), yeasts (e.g. Saccharomyces cerevisiae), filamentous fungi (e.g. Aspergillus spec), plant cells, animal and insect cells. Preferably, the system is mammalian cells, such as CHO cells, available from the ATCC American Type Culture Collection.

A typical mammalian cell vector plasmid for constitutive expression comprises a CMV or SV40 promoter with a suitable poly-A tail and a resistance marker such as neomycin. One example is pSVL, available from Pharmacia, Piscataway, New Jersey, USA. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. Suitable yeast plasmid vectors are pRS403-406 and pRS413-416 and are typically available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integration plasmids (Yips) and include the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are yeast centromere plasmids (Ycp). CMV promoter-based vectors (eg, from Sigma-Aldrich) provide transient or sustained expression, cytoplasmic expression or secretion, and N-terminal or C-terminal tagging in various combinations of FLAG, 3xFLAG, c-myc, or MAT. These fusion proteins allow detection, purification and analysis of the recombinant protein. Double-labeled merges provide flexibility in detection.

A strong regulatory region of the human cytomegalovirus (CMV) promoter increases constitutive protein expression levels reaching 1 mg/L in COS cells. For less active cell lines, protein levels are typically ~0.1 mg/L. The presence of the SV40 origin of replication will result in high levels of DNA replication in permissive COS cells. CMV vectors, for example, may contain the pMB1 origin of replication (a derivative of pBR322) in bacterial cells, a beta-lactamase gene to select for ampicillin resistance in bacteria, human growth hormone polyA, and an f1 origin of replication. Vectors containing a preprotrypsin (PPT) leader sequence can direct secretion of FLAG fusion proteins into culture media for purification using anti-FLAG antibodies, resins and plates. Other vectors and expression systems for use with various host cells are well known in the art.

In another preferred embodiment, two or more peptides or peptide variants of the invention are encoded and thus expressed sequentially (as in a bead-on-a-string structure).

For these purposes, the peptides or peptide variants may be joined or fused together using linker amino acid fragments such as, for example, LLLLLL, or may be joined without any additional peptide(s) between them. These structures could also be used in anticancer therapy and possibly induce immune responses involving both MHC class I and MHC class II molecules.

The present invention also relates to a host cell transformed with the polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. The bacterial cells may preferably be prokaryotic host cells under some conditions and are typically a strain of E. coli, such as, for example, E. coli strain DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1, available from the American Type Culture Collection (ATCC), Rockville, Maryland, USA (ATCC No. 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as lineages fibroblast cells and colon cells from species such as mouse, rat, monkey or human Yeast host cells include YPH499, YPH500 and YPH501, which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA Preferred Mammalian host cells include Chinese hamster ovary (CHO) cells available from ATCC as CCL61, NIH/3T3 Swiss mouse embryonic cells available from ATCC as CRL 1658, monkey kidney COS-1 cells available from ATCC in ATCC as CRL 1650, and cells 293, which are human embryonic kidney cells. Preferred insect cells are Sf9 cells, which can be transfected using baculovirus expression vectors. An overview regarding the selection of suitable host cells for expression is presented, for example, in the study guide by Paulina Balbas and Argelia Lorence Methods in Molecular Biology Recombinant Gene Expression, Reviews and Protocols, Part One, Second Edition, ISBN 978-1-58829-262- 9, and other literature known to one skilled in the art.

Transformation of appropriate host cells using the DNA construct of the present invention is carried out using well known methods, which usually depend on the type of vector used. Regarding transformation of prokaryotic host cells, see, for example, the work of Cohen et al. (Cohen et al., 1972) and (Green and Sambrook, 2012). Transformation of yeast cells is described in the work of Sherman et al. (Sherman et al., 1986). The Beggs method is also suitable (Beggs, 1978). As for vertebrate cells, reagents suitable for transfecting such cells are

- 42 045010 e.g. calcium phosphate and DEAE-dextran or liposomal formulations, available from Stratagene Cloning Systems or Life Technologies Inc., Gaithersburg, MD 20877, USA. Electroporation is also suitable for transforming and/or transfecting cells and is well known in the art for transforming yeast cells, bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain the DNA construct of the present invention can be identified by well known methods such as PCR. Alternatively, the presence of protein in the supernatant can be detected using antibodies.

It should be understood that some host cells of the invention are suitable for producing the peptides of the invention, for example, bacterial, yeast and insect cells. However, other host cells may be used in specific therapeutic modalities. For example, antigen presenting cells, such as dendritic cells, can advantageously be used to express the peptides of the invention so that they can be loaded onto suitable MHC molecules. Thus, the present invention provides a host cell including a nucleic acid or expression vector in accordance with the invention.

In a preferred embodiment, the host cell is an antigen presenting cell, in particular a dendritic cell or an antigen presenting cell. APCs loaded with recombinant prostatic acid phosphatase (PAP) fusion protein were approved by the US Food and Drug Administration (FDA) on April 29, 2010 for use in the treatment of metastatic HRPC (hormone refractory prostate cancer), asymptomatic or with minimal symptoms (sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

Another aspect of the invention provides a method for producing a peptide or a variant thereof, the method comprising culturing a host cell and isolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, nucleic acid or expression vector of the invention is used in medicine. For example, the peptide or a variant thereof may be formulated for intravenous (IV) administration, subcutaneous (SC) administration, intradermal (IC) administration, intraperitoneal (IP) administration, or intramuscular (IM) administration. Preferred routes of administration of the peptides include SC, IV, IP, IM and IV. Preferred methods of administering DNA include iv, i.m., s.c., i.p., and i.v. Doses of 50 μg to 1.5 mg, preferably 125 to 500 μg, of peptide or DNA may be administered, depending on the peptide or DNA concerned. Dosages in this range have been used successfully in previous clinical studies (Walter et al., 2012).

The polynucleotide used in active vaccination may be substantially pure or contained in a suitable vector or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA, or a combination thereof. Methods for constructing and introducing such a nucleic acid are well known in the art. A review is presented, for example, in the work of Teufel et al. (Teufel et al., 2005). Polynucleotide vaccines are easy to produce, but the mechanism of action of these vectors to induce an immune response is not fully understood. Suitable vectors and delivery systems include viral DNA and/or RNA, such as systems that are based on adenovirus, vaccinia virus, retroviruses, herpes virus, adeno-associated virus, or hybrids containing elements of more than one virus. Non-viral delivery systems include cationic lipids and cationic polymers and are well known in the art in the field of DNA delivery. Physical delivery, such as via a gene gun, may also be used. The peptide or peptides encoded by the nucleic acid may be a fusion protein, for example, with an epitope that stimulates T cells against the corresponding opposite complementarity determining region CDR, as described above.

The medicament of the invention may also include one or more adjuvants. Adjuvants are substances that non-specifically enhance or potentiate the immune response (for example, immune responses mediated by CD8-positive T cells or helper T cells (TH) to an antigen, and may thus be considered useful in the medicament of the present invention Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, imiquimod (ALDARA®), resimiquimod, ImuFact IMP321, interleukins such as IL-2, IL-13, IL-21, interferon alpha or beta or their pegylated derivatives, IS Patch, ISS, ISCOMATRIX, ISCOM immunostimulating complexes, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197 -MP-EC, ONTAK, OspA, PepTel® vector system, poly(lactide coglycolide) [PLG] and dextran based microparticles, talactoferrin SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan , Pam3Cys, Aquila QS21 stimon, which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics and other patented adjuvants

- 43 045010 guys, such as Detox from Ribi, Quil or Superfos. Preferred adjuvants are such as Freund's adjuvant or GM-CSF. Several immunological adjuvants (eg, MF59) specific for dendritic cells and their preparation have been described previously (Allison and Krummel, 1995). Cytokines may also be used. Several cytokines have been directly correlated with influencing the migration of dendritic cells to lymphoid tissues (eg, TNF-α), accelerating the maturation of dendritic cells into effective antigen-presenting cells to T cells (eg, GM-CSF, IL-1 and IL-4 ) (US Patent No. 5,849,589, expressly incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23, IL-7, INFalfa, INF-beta) (Gabrilovich et al., 1996).

Immunostimulatory CpG oligonucleotides have also been reported to enhance the effects of vaccine adjuvants. Without wishing to be bound by any particular theory, the authors believe that CpG oligonucleotides act through Toll-like receptors (TLRs), primarily TLR9, to activate the innate (not acquired) immune system. CpG-induced activation of TLR9 enhances antigen-specific humoral and cellular responses to a wide range of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, the maturation and differentiation of dendritic cells is improved, leading to increased activation of TH1 cells and increased production of cytotoxic T lymphocytes (CTLs) even in the absence of assistance from CD4 T cells. TH1 activation caused by TLR9 stimulation persists even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote TH2 activation. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions or similar formulations, which are particularly necessary to initiate a strong response if the antigen is relatively weak. They also speed up the immune response and reduce antigen doses by approximately two orders of magnitude compared to antibody responses to a full dose of non-CpG vaccine, as observed in some experiments (Krieg, 2006). US Pat. No. 6,406,705 B1 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigen to elicit an antigen-specific immune response. The CpG TLR9 antagonist is dSLIM (double-stranded stem-loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules may also be used, such as TLR 7, TLR 8 and/or TLR 9 RNA binding molecules.

Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g., CpR, Idera), ds-RNA analogues such as poly(1:C) and derivatives thereof (e.g., AmpliGen®, Hiltonol®, poly -(ICLC), poly(IC-R), poly(I:C12U), bacterial DNA or RNA other than CpG, and immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab®, Celebrex, NCX-4016 , sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting major structures of the immune system (for example, antibodies to CD40, TGF- beta, TNF-alpha receptor) and SC58175, which can act therapeutically and/or as adjuvants.The amounts and concentrations of adjuvants and additives suitable for use in the context of the present invention can be easily determined by one skilled in the art without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides and derivatives thereof, poly(1:C) and its derivatives, RNA, sildenafil and microsolid formulations with PLG or virosomes.

In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is selected from the group consisting of colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod and interferon-alpha.

In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is selected from the group consisting of colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod and resiquimod. In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvants are Montanide IMS 1312, Montanide ISA206, Montanide ISA 50 V, Montanide ISA-51, poly-ICLC (Hiltonol®) and anti-CD40 monoclonal antibodies or combinations thereof.

This composition is used for parenteral administration, such as subcutaneous, intradermal, intramuscular or oral administration. For this purpose, peptides and - optionally - other molecules

- 44 045010 ly are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. In addition, the composition may contain auxiliary substances such as buffers, coupling agents, ballasts, diluents, flavoring agents, lubricants, etc. The peptides may also be administered together with immunostimulating agents such as cytokines. An extensive list of excipients that can be used in such a composition can be taken, for example, from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The composition can be used for the prevention, prophylaxis and/or treatment of adenomatous or cancerous diseases. Examples of pharmaceutical compositions can be taken, for example, from EP 2112253.

In one aspect, peptides or other molecules described herein may be combined with an aqueous carrier. In one aspect, the aqueous carrier is selected from: ion exchanger, alumina, aluminum stearate, magnesium stearate, lecithin, whey proteins such as human serum albumin, buffering agents such as phosphorates, glycine, sorbic acid, potassium sorbate, mixtures of saturated partial glycerides vegetable fatty acids, salts or electrolytes such as protamine sulfate, disodium phosphate, dicalcium phosphate, dipotassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, polyvinylpyrrolidone vinyl acetate, cellulose-based substances (e.g. microcrystalline cellulose , hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose phthalate), starch, lactose monohydrate, mannitol, trehalose, sodium lauryl sulfate and croscarmellose sodium, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, polymethacrylate, waxes, polyethylene-polyoxypropylene block copolymers, polyethylene glycol and lanolin.

In one aspect, the aqueous carrier contains a plurality of components, such as water, together with a non-aqueous carrier component, such as those described herein. In another aspect, an aqueous carrier is capable of providing improved properties in combination with a peptide or other molecule described herein, such as improved solubility, potency, and/or improved immunotherapeutic properties. In addition, the composition may contain auxiliary substances such as buffers, coupling agents, ballasts, diluents, flavoring agents, lubricants, etc. The pharmaceutically acceptable diluent, for example, may include solvents, bulking agents, stabilizers, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, having physiological compatibility. Examples of pharmaceutically acceptable diluents include one or more of saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases it will be preferable to include one or more isotonic substances, for example sugars such as trehalose and sucrose, polyhydric alcohols such as mannitol, sorbitol or sodium chloride. Pharmaceutically acceptable substances such as wetting agents or small amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers are also included within the scope of the present invention. In addition, the composition may contain auxiliary substances such as buffers, coupling agents, ballasts, diluents, flavoring agents and lubricants.

It is important to understand that the immune response induced by the vaccine according to the invention is directed to cancer cells at different stages of the cell cycle and different stages of tumor development. In addition, the attack targets various signaling pathways associated with cancer. This is an advantage over vaccines targeting only one or a few targets, which may result in the tumor easily adapting to such an attack (tumor escape). In addition, not all individual tumors have the same antigen expression patterns. Therefore, the combination of several tumor-associated peptides ensures that each individual tumor has at least some of these targets. The composition is designed based on the fact that each tumor is expected to express multiple antigens and span multiple independent signaling pathways required for tumor growth and maintenance. Thus, the ready-to-use vaccine can be easily used in a larger patient population. This means that pre-selection of patients for vaccine treatment can be limited by HLA typing, without requiring any additional analysis of biomarkers of antigen expression, but still ensures that multiple targets of the induced immune response are simultaneously targeted, which is important for efficacy (Banchereau et al ., 2001; Walter et al., 2012).

As used herein, the term scaffold refers to a molecule that specifically binds to a (eg, antigenic) determinant. In one embodiment, the scaffold is capable of directing the unit to which it is attached (e.g., a (second) antigen-binding element) to a target site, e.g., a specific type of tumor cell or tumor stroma bearing an antigenic determinant (e.g., a peptide-MHC complex according to this patent application). In another embodiment, the scaffold is capable of activating signaling pathways by

- 45 045010 its target antigen, for example, T-cell receptor complex antigen. Scaffolds include, but are not limited to, antibodies and fragments thereof, antibody antigen binding domains including an antibody heavy chain variable region and an antibody light chain variable region, binding proteins including at least one ankyrin repeat motif and single domain antigen binding (SDAB) molecules, aptamers, (soluble) TCRs and (modified) cells such as allogeneic or autologous T cells. To assess whether a molecule is a scaffold that binds to a target, a binding assay can be performed.

Specific binding means that the scaffold binds to the peptide-MHC complex of interest better than to other naturally occurring peptide-MHC complexes to such an extent that the scaffold, equipped with an active molecule capable of killing a cell bearing a specific target, is unable to kill another a cell without a specific target, but presenting another complex(es) of the peptide with MHC. Binding to other peptide-MHC complexes does not play a role if the peptide of the cross-reacting peptide-MHC complex is not naturally occurring, i.e. not derived from the human HLA peptidome. Tests to evaluate target cell killing potential are well known in the art. They should be performed using target cells (primary cells or cell lines) with intact presentation of peptide-MHC complexes or cells loaded with peptides such that levels of naturally occurring peptide-MHC complexes are achieved.

Each scaffold may include a tag that enables detection of an associated scaffold by determining the presence or absence of a signal provided by the tag. For example, the scaffold may be labeled with a fluorescent dye or any other suitable cell marker molecule. Such marker molecules are well known in the art. For example, fluorescent labeling, such as with a fluorescent dye, can allow visualization of the bound aptamer by fluorescence or laser scanning microscopy or flow cytometry.

Each scaffold may be conjugated to a second active molecule, such as, for example, IL-21, an anti-CD3 antibody, and an anti-CD28 antibody.

For more information on polypeptide scaffolds, see, for example, the prior art section of patent application WO 2014/071978 A1 and the literature cited therein.

The present invention further relates to aptamers. Aptamers (see, for example, WO 2014/191359 and the literature cited therein) are short, single-stranded nucleic acid molecules that can fold into specific three-dimensional structures and recognize specific target structures. They have proven to be a suitable alternative for the development of targeted therapies. Aptamers have been demonstrated to selectively bind to a variety of complex targets with high affinity and specificity.

Aptamers that recognize molecules found on the surface of cells have been identified in the last decade and provide an opportunity for the development of diagnostic and therapeutic approaches. Since aptamers have been demonstrated to have little to no toxicity or immunogenicity, they are promising candidates for biomedical applications. Indeed, aptamers, such as those recognizing prostate-specific membrane antigen, have been successfully used in targeted therapy and have demonstrated functionality in in vivo xenograft models. In addition, aptamers that recognize specific tumor lines have been identified.

DNA aptamers can be selected that exhibit a wide range of properties for recognizing various cancer cells, and in particular, cells derived from solid tumors, while non-tumorigenic and primary healthy cells are not recognized. If the identified aptamers recognize not only a specific tumor subtype, but also interact with various tumors, this makes it possible to use aptamers as so-called broad-spectrum diagnostic and therapeutic agents.

Moreover, the study of cell binding behavior using flow cytometry showed that the aptamers exhibited very good apparent affinity, which was expressed at the nanomolar level.

Aptamers are suitable for diagnostic and therapeutic purposes. Additionally, some aptamers could be demonstrated to be taken up by tumor cells and thus could act as molecular carriers to target anticancer agents such as siRNA to tumor cells.

Aptamers can be selected for complex targets such as cells and tissues and peptide complexes comprising, preferably consisting of a sequence in accordance with any of the sequences of SEQ ID NO 1 to SEQ ID NO 489, in accordance with the present invention with an MHC molecule using cell-SELEX method (Systematic Evolution of Ligands by Exponential enrichment - systematic evolution of ligands with exponential enrichment).

The peptides of the present invention can be used for the production and development of specific antibodies to MHC/peptide complexes. They can be used in therapies that target toxins or radioactive substances to affected tissue. Another use of these antibodies could be to target radionuclides to diseased tissue for imaging purposes such as PET (positron emission tomography). This can help in detecting small metastases or in determining the size and exact location of affected tissue.

Thus, in another aspect of the invention, there is provided a method for producing a recombinant antibody that specifically binds to human major histocompatibility complex (MHC) class I or II in complex with an HLA-restricted antigen (preferably a peptide in accordance with the present invention), the method comprising: immunization genetically a modified non-human mammal containing cells expressing molecules of the specified human major histocompatibility complex (MHC) class I or II with a soluble form of the MHC class I or II molecule in complex with the specified HLA-restricted antigen; isolating mRNA molecules from antibody-producing cells of said non-human mammal; creating a phage display library containing phages displaying protein molecules encoded by said mRNA molecules; and isolating at least one phage from said phage display library, wherein said at least one phage displays on the surface said antibody that specifically binds to said human major histocompatibility complex (MHC) class I or II in complex with said HLA-restricted antigen.

Another aspect of the invention therefore provides an antibody that specifically binds to human major histocompatibility complex (MHC) class I or II in complex with an HLA-restricted antigen, where the antibody is preferably a polyclonal antibody, a monoclonal antibody, a bispecific antibody and/or a chimeric antibody. antibody.

Appropriate methods for producing such antibodies and single-chain major histocompatibility complex class I, as well as other tools for producing these antibodies, are disclosed in patent applications WO 03/068201, WO 2004/084798, WO 01/72768, WO 03/070752 and in published works (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), all of which for the purposes of the present invention are expressly incorporated by reference in their entirety.

Preferably, the antibody binds with a binding affinity of less than 20 nanomoles, preferably less than 10 nanomoles, to the complex, which is also referred to as specific in the context of the present invention.

The present invention provides a peptide comprising a sequence that is selected from the group consisting of the sequences of SEQ ID NO 1 through SEQ ID NO 489, or a variant thereof that is at least 88% homologous (preferably identical) to the sequence of SEQ ID NO 1 according to SEQ ID NO 489, or a variant thereof, which induces a cross-reaction of T cells with the specified peptide, where the specified peptide is not a full-length basic polypeptide.

The present invention further provides a peptide comprising a sequence that is selected from the group consisting of the sequences of SEQ ID NO 1 through SEQ ID NO 489, or a variant thereof that is at least 88% homologous (preferably identical) to SEQ ID NO 1 according to SEQ ID NO 489, wherein the peptide or variant thereof has a total length of from 8 to 100, preferably from 8 to 30, and most preferably from 8 to 14 amino acids.

The present invention further relates to peptides in accordance with the invention capable of binding to a human major histocompatibility complex (MHC) class I or II molecule.

The present invention further relates to peptides in accordance with the invention, where the peptide consists of or consists essentially of an amino acid sequence in accordance with SEQ ID No. 1 to SEQ ID No. 489.

The present invention further relates to peptides in accordance with the invention, where the peptide is modified (chemically) and/or includes non-peptide bonds.

The present invention further relates to peptides in accordance with the invention, where the peptide is part of a fusion protein, in particular comprising the N-terminal amino acids of the HLA-DR antigen-associated invariant chain (li), or where the peptide is fused to an antibody (or fused to an antibody sequence) , for example, an antibody that is specific for dendritic cells.

The present invention further relates to nucleic acid encoding peptides in accordance with the invention, provided that the peptide is not entirely human protein.

The present invention further relates to a nucleic acid according to the invention, which is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable of expressing a nucleic acid in accordance with the present invention.

The present invention further relates to a peptide in accordance with the present invention, a nucleic acid in accordance with the present invention, or an expression vector in accordance with the present invention for use in medicine, in particular in the treatment of lung cancer (including NSCLC and SCLC).

- 47 045010

The present invention further relates to a host cell comprising a nucleic acid in accordance with the present invention or an expression vector in accordance with the present invention.

The present invention further relates to a host cell according to the present invention, which is an antigen presenting cell, preferably a dendritic cell.

The present invention further relates to a method for producing a peptide in accordance with the present invention, the method comprising culturing a host cell in accordance with the present invention and isolating the peptide from said host cell or its culture medium.

The present invention further relates to a method in accordance with the present invention, wherein the antigen is loaded onto MHC class I or II molecules expressed on the surface of a suitable antigen presenting cell upon contact of a sufficient amount of the antigen with the antigen presenting cell.

The present invention further relates to a method according to the invention, wherein the antigen presenting cell includes an expression vector capable of expressing said peptide containing the sequence of SEQ ID No. 1 to SEQ ID No. 489 or the specified variant amino acid sequence.

The present invention further relates to activated T cells produced by a method in accordance with the present invention, wherein said T cells selectively recognize a cell that aberrantly expresses a polypeptide comprising an amino acid sequence in accordance with the present invention.

The present invention further relates to a method of killing target cells in a patient whose target cells aberrantly express a polypeptide comprising any amino acid sequence in accordance with the present invention, the method comprising administering to the patient an effective number of T cells in accordance with the present invention.

The present invention further relates to the use of any described peptide, nucleic acid in accordance with the present invention, expression vector in accordance with the present invention, cell in accordance with the present invention or activated cytotoxic T lymphocyte in accordance with the present invention as a medicament or in the manufacture of a medicament . The present invention further relates to a method of administration according to the present invention, wherein the medicament exhibits anti-cancer activity.

The present invention further relates to a method of administration according to the invention, wherein the medicament is a vaccine. The present invention further relates to a method of administration according to the invention, wherein the medicament exhibits anticancer activity.

The present invention further relates to uses in accordance with the invention, wherein said cancer cells are lung cancer cells (including NSCLC and SCLC) or other solid or hematological tumor cells, such as acute myeloid leukemia cells, breast cancer, bile duct cancer, brain cancer, chronic lymphocytic leukemia, colorectal carcinoma, esophageal cancer, gallbladder cancer, stomach cancer, squamous cell carcinoma of the head and neck, hepatocellular cancer, melanoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, cancer bladder, uterine cancer.

The present invention further relates to specific protein markers and peptide-based biomarkers in accordance with the present invention, in the context of the invention called targets, which can be used in making a diagnosis and/or prognosis of lung cancer (including NSCLC and SCLC). The present invention also relates to the use of these new targets for the treatment of cancer.

The term antibody or antibodies is used in the context of this invention in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact or complete immunoglobulin molecules, the term antibody also includes fragments (eg, CDRs, Fv, Fab and Fc fragments) or polymers of such immunoglobulin molecules and humanized versions of immunoglobulin molecules, provided that they exhibit any of the desired properties (eg , specifically bind to a (poly)peptide marker of lung cancer (including NSCLC and SCLC), deliver the toxin to a lung cancer cell (including NSCLC and SCLC) expressing the cancer marker gene at an increased level and/or inhibit the activity of the polypeptide - marker of lung cancer (including NSCLC and SCLC)) in accordance with the present invention.

If possible, the antibodies of the invention can be purchased from commercial sources. Antibodies of the invention can also be prepared using well known methods. An experienced specialist will understand that both full-length polypeptide markers of lung cancer (including NSCLC and SCLC) and their fragments can be used to obtain antibodies according to the invention. The polypeptide necessary to produce the antibody of the invention may be partially or completely purified from a natural source or may be produced using recombinant DNA techniques.

- 48 045010

For example, a cDNA encoding a peptide of the present invention, such as the peptide of SEQ ID NO: 1 to SEQ ID NO: 489, a polypeptide or variant or fragment thereof, can be expressed in prokaryotic cells (eg, bacteria) or eukaryotic cells (e.g., yeast, insect or mammalian cells), after which the recombinant protein can be purified and used in the production of a monoclonal or polyclonal antibody preparation that specifically binds to the polypeptide marker of lung cancer (including NSCLC and SCLC) used to obtain the antibody according to the invention.

One of ordinary skill in the art will appreciate that producing two or more different sets of monoclonal or polyclonal antibodies increases the likelihood of obtaining an antibody with the specificity and affinity required for its intended use (eg, ELISA, immunohistochemistry, in vivo imaging, immunotoxin-based therapy). Antibodies are tested for their desired activity using known methods in accordance with the purpose of the antibody (e.g. ELISA, immunohistochemistry, immunotherapy, etc.; for further information on generating and testing antibodies see, for example, Greenfield, 2014). , 2014)). For example, antibodies can be tested by ELISA or Western blot, immunohistochemical staining of formaldehyde-fixed cancer tissue samples or frozen tissue sections. After initial characterization in vitro, antibodies intended for therapeutic or diagnostic use in vivo are examined in accordance with known clinical research methods.

The term monoclonal antibody in the context of the present invention means an antibody obtained from an essentially homogeneous population of antibodies, i.e. individual antibodies within a population are identical except for possible natural mutations that may be present in small quantities. Monoclonal antibodies in the context of the present invention specifically include chimeric antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequences of antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remaining portion(s) i) the chain is identical(s) or homologous(es) to the corresponding sequences of antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity (US Patent No. 4,816 567, which is included herein in its entirety).

Monoclonal antibodies according to the invention can be obtained using the hybridoma method. In the hybridoma technique, a mouse or other suitable host animal is typically immunized with an immunizing agent to initiate lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

Monoclonal antibodies can also be produced using recombinant DNA technologies, such as those described in US Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be easily isolated and sequenced using standard techniques (for example, using oligonucleotide probes that able to specifically bind to genes encoding the heavy and light chains of mouse antibodies).

In vitro methods are also suitable for the production of monovalent antibodies. Cleavage of antibodies to obtain their fragments, especially Fab fragments, can be done using standard techniques known in the art. For example, cleavage can be carried out using papain. Examples of cleavage by papain are described in WO 94/29348 and US Patent No. 4,342,566. Cleavage of antibodies by papain typically results in two identical antigen-binding fragments called Fab fragments, each having a distinct antigen-binding site and residual Fc fragment. As a result of treatment with pepsin, the F(ab') ₂ fragment and the pFc' fragment are obtained.

Antibody fragments, whether linked or unlinked to other sequences, may also include insertions, deletions, substitutions, or other selected modifications of specific regions or amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to an unmodified antibody or antibody fragment. These modifications can introduce some additional properties, such as adding/removing amino acids capable of disulfide binding, increasing their biological stability, changing their secretory characteristics, etc. In any case, the antibody fragment must have the property of biological activity, such as binding activity, regulation of binding on the binding domain, etc. The functional or active regions of an antibody can be identified by mutagenesis of a specific region of the protein, followed by expression and examination of the expressed polypeptide. Such methods will be readily apparent to those skilled in the art and may involve site-directed mutagenesis of the nucleic acid encoding the antibody fragment.

Antibodies of the invention may further include humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immu

- 49 045010 noglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab' or other antigen-binding antibody subsequences) that contain minimal sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from the complementary determinant regions (CDRs) of the recipient are replaced with residues from the CDRs of a non-human species (donor antibody), such as mice, rats or rabbits, having the desired specificity, affinity and binding capacity. In some cases, Fv-framework (FR) residues of human immunoglobulin are replaced by corresponding residues of non-human origin. Humanized antibodies may also include residues that are not found in either the recipient antibody or the imported CDR or framework sequences. Typically, a humanized antibody will include substantially all of at least one and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions correspond to those of the consensus human immunoglobulin sequences. Optimally, the humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. Such amino acid residues of non-human origin are often referred to as imported residues, which are usually taken from the imported variable domain. Humanization can be substantially accomplished by replacing rodent CDR regions or CDR sequences with corresponding human antibody sequences. Accordingly, such humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567) where substantially less than one intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly FR residues have been replaced with similar site residues from rodent antibodies.

Transgenic animals (for example, mice) that are capable of producing the full spectrum of human antibodies during immunization in the absence of endogenous immunoglobulin production can be used. For example, homozygous deletion of the gene encoding the antibody heavy chain attachment region in chimeric and germline mutant mice has been described to result in complete inhibition of endogenous antibody production. Transfer of a human germline immunoglobulin gene template into such germline mutant mice will result in the production of human antibodies following antigen stimulation. Human antibodies can also be produced in phage display libraries.

The antibodies of the invention are preferably administered to a subject in a pharmaceutically acceptable carrier. A suitable amount of a pharmaceutically acceptable salt is typically used in the formulation to render the composition isotonic. Examples of pharmaceutically acceptable carriers include saline solution, Ringer's solution and glucose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. In addition, carriers are provided that include sustained release formulations, such as semi-permeable matrices of solid hydrophobic polymers containing the antibody, the matrices of which are in the form of shaped objects, for example, films, liposomes or microparticles. It will be apparent to one skilled in the art that certain carriers may be more preferred depending on, for example, the route of administration and the concentration of the antibody administered.

Antibodies can be administered to a subject, patient, or cell by injection (eg, intravenous, intraperitoneal, subcutaneous, intramuscular) or other means, such as infusion, which ensures delivery to the bloodstream in an efficient manner. Antibodies can also be administered by intratumoral or peritumoral routes to produce local as well as systemic therapeutic effects. Local or intravenous administration is preferred.

The effective dosage and mode of administration of antibodies can be determined empirically, and such decisions can be made by a specialist in the field. Those skilled in the art will appreciate that the dosage of antibodies to be administered will vary depending on, for example, the subject to whom the antibody will be administered, the route of administration, the particular type of antibody used, and other medications administered. A typical daily dose of antibodies for antibody monotherapy may vary from about 1 μg/kg up to 100 mg/kg body weight or more per day, depending on the factors mentioned above. Following administration of an antibody, preferably for the treatment of lung cancer (including NSCLC and SCLC), the effectiveness of the therapeutic antibody can be assessed by various methods known to one of ordinary skill in the art. For example, the size, number and/or distribution of cancer in a subject undergoing treatment can be monitored using standard tumor imaging techniques. An antibody administered for therapeutic purposes that blocks tumor growth, causes the tumor to shrink in size and/or prevents the development of new tumors over the course of the disease that would have occurred without the antibody, is an effective antibody for treating cancer.

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Another aspect of the invention provides a method for producing a soluble T-cell receptor (TCR) that recognizes a specific peptide-MHC complex. Such soluble T cell receptors can be derived from specific T cell clones, and their affinity can be increased by mutagenesis targeting complementarity determining regions. Phage display can be used to select a T cell receptor (US application 2010/0113300, (Liddy et al., 2012)). In order to stabilize T cell receptors during phage display and in practical drug applications, the alpha and beta chains can be linked, for example through non-naturally occurring disulfide bonds, other covalent bonds (single chain T cell receptor) or by dimerization domains (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999). In order to perform certain functions on target cells, the T cell receptor may be associated with toxins, drugs, cytokines (see, for example, US application 2013/0115191) and effector cell recruitment domains, such as the anti-CD3 domain, etc. d. Moreover, it can be expressed on T cells used for adoptive transfer. Further information can be found in patent applications WO 2004/033685A1 and WO 2004/074322 A1. The combination of soluble TCRs is described in patent application WO 2012/056407 A1. Other production methods are described in patent application WO 2013/057586 A1.

In addition, the peptides and/or TCRs or antibodies or other binding molecules of the present invention can be used to confirm a diagnosis of cancer made by a pathologist based on examination of a biopsy specimen.

These antibodies or TCRs can also be used for in vivo diagnostics. Typically, the antibody is labeled with a radionucleotide (such as ⁿ¹ In, ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ³ H, ³² P or ³⁵ S) so that the tumor can be localized by immunoscintigraphy. In one embodiment, antibodies or fragments thereof bind to the extracellular domains of two or more protein targets selected from the group consisting of the above proteins with an affinity (Kd) of less than 1x10 μM.

Antibodies for diagnostic purposes can be labeled with probes suitable for detection by various imaging modalities. Probe detection methods include, but are not limited to, fluorescence, light, confocal and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium and other lanthanides, paramagnetic iron, fluorine-18 and other positron-active radionuclides. Moreover, probes can be bi- or multi-functional and can be detected by more than one of the above methods. These antibodies can be labeled directly or indirectly with these probes. Attachment of probes to antibodies includes covalent attachment of the probe, insertion of the probe into the antibody, and covalent attachment of a chelating compound to attach the probe, among other well-established techniques in the art. For immunohistochemical studies, the affected tissue sample may be fresh or frozen, or may be embedded in paraffin and fixed with a preservative such as formalin. The fixed or embedded section is brought into contact with a labeled primary antibody and a secondary antibody, where the antibody is used to detect in situ protein expression.

Another aspect of the present invention includes a method for producing activated T cells in vitro, the method comprising contacting the T cells in vitro with antigen-loaded human MHC molecules expressed on the surface of a suitable antigen presenting cell for a period of time sufficient to activate the T cell in an antigen-specific manner wherein the antigen is a peptide according to the invention. Preferably, a sufficient amount of antigen is used with the antigen presenting cell.

Preferably, the mammalian cell does not have the TAP peptide transporter or has a reduced level or reduced functional activity. Suitable cells deficient in the TAP peptide transporter include T2, RMA-S and Drosophila cells. TAP is a transporter associated with antigen processing.

The T2-deficient human cell line loaded with the peptides is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA under catalog number CRL 1992; Drosophila cell line, Schneider line 2 is available from ATCC under catalog number CRL 19863; the mouse RMA-S cell line is described by Ljunggren et al. (Ljunggren and Karre, 1985).

Preferably, prior to transfection, said host cell does not substantially express MHC class I molecules. It is also preferred that the stimulator cell expresses a molecule important in providing a costimulatory signal to T cells, such as any of B7.1, B7.2, ICAM-1 and LFA 3. Nucleic acid sequences of numerous MHC class I molecules and costimulatory molecules publicly available in GenBank and EMBL databanks.

When using an MHC class I epitope as an antigen, the T cells are CD8 positive T cells.

If an antigen presenting cell is transfected to express such an epitope, then

- 51 045010 It is desirable that the cell includes an expression vector capable of expressing the peptide containing SEQ ID No. 1 to SEQ ID No. 489 or a variant of such amino acid sequence.

Many other methods can be used to obtain T cells in vitro. For example, autologous tumor-infiltrating lymphocytes are used to generate CTLs. Plebanski et al. (Plebanski et al., 1995) used autologous peripheral blood lymphocytes (PBL) to obtain T cells. In addition, it is possible to obtain autologous T cells by loading dendritic cells with a peptide or polypeptide or by infection with a recombinant virus. B cells can also be used to generate autologous T cells. In addition, macrophages loaded with a peptide or polypeptide or infected with a recombinant virus can be used to obtain autologous T cells. S. Walter et al. (Walter et al., 2003) describe in vitro priming of T cells using artificial antigen presenting cells (iAPCs), which is also a suitable way to generate T cells against a selected peptide. In the present invention, iAPCs were obtained by attaching preformed MHC peptide complexes to the surface of polystyrene particles (microspheres) using a biochemical biotin-streptavidin method. This system allows precise control of MHC density on iAPCs, which allows the selective induction of high- or low-avidity antigen-specific T-cell responses with high efficiency in blood samples. In addition to MHC-peptide complexes, iAPCs must carry other proteins with costimulatory activity, such as anti-CD28 antibodies, attached to their surface. In addition, such an iAPC-based system often requires the addition of appropriate soluble factors, for example, cytokines such as interleukin-12.

Allogeneic cells can also be used in the production of T cells, and this method is described in detail in patent application WO 97/26328, incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells can be used for antigen presentation, such as Chinese hamster ovary (CHO) cells, baculovirus-infected insect cells, bacteria, yeast, and vaccinia-infected target cells. In addition, plant viruses can be used (see, for example, Porta et al. (1994), which describes the development of Chinese cowpea mosaic virus as a highly productive system for presenting foreign peptides.

Activated T cells that are directed against the peptides of the invention are useful for therapy. Thus, another aspect of the invention provides activated T cells produced by the above-mentioned methods of the invention.

Activated T cells produced by the above method will selectively recognize a cell that aberrantly expresses a polypeptide comprising the amino acid sequence of SEQ ID No. 1 to SEQ ID NO489.

Preferably, the T cell recognizes the cell upon interaction through its TCR with the HLA/peptide complex (eg, upon binding). The T cells are suitable for a method of killing target cells in a patient whose target cells aberrantly express a polypeptide comprising an amino acid sequence of the invention, wherein an effective number of activated T cells is administered to the patient. T cells that are administered to a patient may be derived from the patient and activated as described above (ie, they are autologous T cells). Alternatively, the T cells are obtained from a person other than the patient. Of course, it is preferable if the individual is a healthy individual. By healthy individual, we mean that the individual is in good general health, preferably has a competent immune system, and more preferably does not suffer from any disease that can be easily monitored and identified.

The in vivo target cells for CD8-positive T cells in accordance with the present invention may be tumor cells (which sometimes express MHC class II molecules) and/or stromal cells surrounding the tumor (tumor cells) (which sometimes also express MHC II molecules class (Dengjel et al., 2006)).

The T cells of the present invention can be used as active ingredients in a therapeutic composition. Thus, the invention also provides a method of killing target cells in a patient whose target cells aberrantly express a polypeptide comprising an amino acid sequence of the invention, the method comprising administering to the patient an effective number of T cells as defined above.

By aberrantly expressed, we also mean that the polypeptide is expressed in excess of expression levels in normal tissues, or that the gene is silent in the tissue of origin of the tumor but is expressed in the tumor. By overexpressed, we mean that the polypeptide is present at a level that is at least 1.2 times higher than the level present in normal tissue; preferably at least 2 times and more preferably at least 4 or 6 times the level present in normal tissue.

T cells can be obtained by methods known in the art, for example, those described above.

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Protocols for this so-called adoptive transfer of T cells are well known in the art. Reviews can be found in Gattioni et al. and Morgan et al. (Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention involves the use of peptides in complex with MHC to produce a T cell receptor, the nucleic acid of which is cloned and introduced into a host cell, preferably a T cell. This engineered T cell can then be administered to a patient to treat cancer.

Any molecule according to the invention, i.e. a peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T cell receptor, or nucleic acid encoding the same is useful for treating disorders characterized by cells that evade the immune response. Therefore, any molecule of the present invention can be used as a medicine or in the production of a medicine. The molecule may be used alone or in combination with other molecule(s) of the invention or known molecule(s).

The present invention also provides a kit comprising:

(a) a container that contains a pharmaceutical composition as described above, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstituting solution for the lyophilized formulation; and (c) optionally, instructions for (i) using the solution or (ii) reconstituting the solution and/or using the lyophilized formulation.

In addition, the kit may also include one or more (iii) buffers, (iv) diluents, (v) filters, (vi) needles, or (v) syringes. The container is preferably a bottle, vial, syringe or tube; and it can be a reusable container. The pharmaceutical composition is preferably lyophilized.

The kit according to the present invention preferably includes a lyophilized composition of the present invention in a suitable container and instructions for its reconstitution and/or for its use. Suitable containers include, for example, bottles, vials (eg, dual chamber vials), syringes (such as dual chamber syringes), and tubes. The container can be made of different materials, such as glass or plastic. Preferably, the kit and/or container contains instructions for use of the container or instructions associated therewith that provide directions for reconstitution and/or use. For example, the label may indicate that the lyophilized formulation must be reconstituted to the peptide concentrations described above. The label may further indicate that the composition is for use or intended for subcutaneous administration.

The formulation container may be a refillable vial that allows repeated administration (eg, 2 to 6 administrations) of the reconstituted formulation. The kit may further include a second container containing a suitable diluent (eg, sodium bicarbonate solution).

After mixing the diluent and the lyophilized formulation, the final concentration of peptide in the reconstituted formulation is preferably at least 0.15 mg/ml/peptide (=75 μg) and preferably no more than 3 mg/ml/peptide (=1500 μg). The kit may further include other materials commercially and user-desirable, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The kits of the present invention may include a single container that contains a dosage form of the pharmaceutical compositions of the present invention with or without other components (eg, other compounds or pharmaceutical compositions of these other compounds) or may have separate containers for each component.

The kit of the invention preferably includes a composition of the invention packaged for use in combination with co-administration of a second compound (such as an adjuvant (eg, GM-CSF), a chemotherapeutic agent, a natural product, a hormone or an antagonist, an anti-angiogenesis agent or an angiogenesis inhibitor; an apoptosis- inducing agent or chelator) or a pharmaceutical composition thereof. The components of the kit may be premixed prior to administration to the patient, or each component may be contained in a separate container. The components of the kit may be provided in the form of one or more liquid solutions, preferably an aqueous solution, more preferably a sterile aqueous solution. The components of the kit may also be provided in solid form, which can be converted into a liquid by adding suitable solvents, which are preferably provided in another, separate container.

The therapeutic kit container may be a vial, test tube, flask, bottle, syringe, or any other means containing a solid or liquid. Typically, if there is more than one component, the kit contains a second vial or other container to allow separate administration. The kit may also contain another container for a pharmaceutically acceptable liquid. The treatment kit will preferably comprise an apparatus (for example, one or

- 53 045010 more needles, syringes, eye droppers, pipettes, etc.), which ensures the administration of the substances according to the invention, which are components of the present kit.

The present composition is suitable for administering the peptides by any suitable route, such as oral (enteral), nasal, ocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal. Preferably, administration is subcutaneous and, most preferably, intravenous administration using an infusion pump.

Since the peptides of the invention have been isolated from lung cancer cells (including NSCLC and SCLC), the medicament of the invention is preferably used for the treatment of lung cancer (including NSCLC and SCLC).

In addition, the present invention further relates to a method for producing a personalized pharmaceutical preparation for an individual patient, comprising producing a pharmaceutical composition comprising at least one peptide selected from the TUMAP repository of pre-screened peptides, wherein the at least one peptide used in the pharmaceutical composition, selected for its suitability for the individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method can be adapted to obtain T cell clones for further use, for example, in the isolation of TCR or soluble antibodies or other treatments.

A personalized pharmaceutical refers to therapeutics designed specifically for an individual patient that will be used exclusively to treat that patient, including actively personalized cancer vaccines and adoptive cell therapies using the patient's autologous tissue.

In the context of the present invention, the term repository refers to a group or set of peptides that have previously been screened for immunogenicity and/or over-presentation in a particular tumor type. The concept of storage does not imply that the specific peptides included in the vaccine were prepared in advance and stored in an actual facility, although this possibility is also taken into account. It is certainly appreciated that the peptides may be produced de novo for each customized vaccine produced, or may be prepared in advance and stored. The repository (eg, in the form of a data bank) consists of tumor-associated peptides that are highly overexpressed in tumor tissue of patients with lung cancer (including NSCLC and SCLC) with various HLA-A, HLA-B and HLAC alleles. It may contain peptides associated with MHC class I and MHC class II molecules or extended peptides associated with MHC class I molecules. In addition to tumor-associated peptides collected from several lung cancer tissues (including NSCLC and SCLC), the repository may contain marker peptides HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*24, HLA- B*07, HLA-B*08 and HLAB*44. These peptides allow a quantitative comparison of the intensity of the T cell immune response induced by TUMAP peptides and therefore provide important insight into the vaccine's ability to induce antitumor responses. Second, they serve as important non-self-antigen-derived positive control peptides in the event that the patient does not exhibit vaccine-induced T-cell responses to self-antigen-derived TUMAP peptides. And third, it may allow inferences to be made regarding the patient's immunocompetence status.

TUMAP peptides for the repository were identified using an integrated functional genomics approach combining gene expression analysis, mass spectrometry and T-cell immunology (XPresident®). This approach ensures that only those TUMAP peptides that are actually present in a high percentage of tumors but are not or only minimally expressed on normal tissue are selected for subsequent analysis. For the initial selection of peptides, lung cancer tissue samples (including NSCLC and SCLC) from patients and blood from healthy donors were analyzed in a stepwise manner.

1. HLA ligands from malignant material were identified using mass spectrometry.

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis was used to identify overexpressed genes in malignant tissue (lung cancer (including NSCLC and SCLC)) compared with a number of normal organs and tissues.

3. Identified HLA ligands were compared with gene expression data. Peptides that are overexpressed or selectively presented on tumor tissue, preferentially encoded by the selectively expressed or overexpressed genes identified in step 2, were considered suitable TUMAP multipeptide vaccine candidates.

4. The literature was reviewed to identify additional evidence supporting the relevance of the peptides identified as TUMAP.

5. The relevance of overexpression at the mRNA level was confirmed by the repeated detection of TUMAP peptides selected in step 3 on tumor tissue and absence (or infrequent detection) on healthy tissues.

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6. To evaluate whether in vivo induction of T cell responses by the selected peptides could be feasible, in vitro immunogenicity assays were performed using human T cells from healthy donors as well as patients with lung cancer (including NSCLC and SCLC).

In one aspect, the peptides are pre-screened for immunogenicity prior to their inclusion in the repository. By way of example, and not as a limitation of the invention, the immunogenicity of the peptides included in the repository is determined by a method involving in vitro T cell priming through repeated stimulation of CD8+ T cells from healthy donors with artificial antigen presenting cells loaded with peptide-MHC complexes and antibodies to CD28.

This method is preferred for rare cancers and patients with rare expression profiles. In contrast to multipeptide cocktails with a constant composition, which have already been developed at this time, the repository makes it possible to achieve a significantly higher correspondence between the actual expression of antigens in the tumor and the composition of the vaccine. Selected individual peptides or combinations of several ready-to-use peptides will be used for each patient in a multi-targeted approach. Theoretically, an approach based on selecting, for example, 5 different antigenic peptides from a library of 50 copies would already lead to approximately 17 million possible drug product formulations (DPs).

In one aspect, peptides for inclusion in a vaccine are selected based on their suitability for an individual patient based on the method of the present invention, as described herein or as set forth below.

HLA phenotype, transcriptomics, and proteomics data are collected from tumor material and patient blood samples to identify the most appropriate peptides for each patient, which includes both TUMAP peptides from the repository and those unique to the patient (i.e., mutated). The peptides selected will be those that are selectively or overexpressed in patient tumors and, where possible, exhibit strong in vitro immunogenicity when assayed with individual patient PBMCs.

Preferably, the peptides included in the vaccine are identified by a method comprising: (a) identification of tumor associated peptides (TUMAP) presented by an individual patient's tumor sample; (b) comparing the peptides identified in step (a) with a repository (data bank) of peptides, as described above; and (c) selecting at least one peptide from a repository that correlates with a tumor-associated peptide identified in the patient. For example, TUMAP peptides presented by a tumor sample are identified by: (a1) comparing expression data in the tumor sample with data from normal tissue corresponding to the tissue type of the tumor sample to identify proteins that are overexpressed or aberrantly expressed in the tumor sample; and (a2) establishing a correlation between the expression data and the MHC ligand sequences associated with class I and/or II MHC molecules in the tumor sample to identify MHC ligands that are derived from proteins overexpressed or aberrantly expressed by the tumor. Preferably, MHC ligand sequences are identified by eluting bound peptides from MHC molecules isolated from a tumor sample and sequencing the eluted ligands. Preferably, the tumor sample and normal tissue are obtained from the same patient.

Additionally, or as an alternative to this, by selecting peptides using a repository model, TUMAP peptides can be identified de novo from a patient and then included in a vaccine. As one example, candidate TUMAP peptides can be identified in a patient by (a1) comparing expression data in a tumor sample with normal tissue data corresponding to the tissue type of the tumor sample to identify proteins that are overexpressed or aberrantly expressed in the tumor sample ; and (a2) establishing a correlation between the expression data and the sequences of MHC ligands bound to class I and/or II MHC molecules in the tumor sample to identify MHC ligands that are derived from proteins overexpressed or aberrantly expressed by the tumor. As another example, proteins having mutations that are unique to a tumor sample matched to the corresponding normal tissue of an individual patient can be identified, and TUMAP peptides that specifically target the mutation can be identified. For example, the genome of a tumor and corresponding normal tissue can be sequenced using whole-genome sequencing: to detect nonsynonymous mutations in protein-coding regions of genes, genomic DNA and RNA are extracted from tumor tissues, and normal, mutation-free germline genomic DNA is extracted from peripheral blood mononuclear cells ( IPC). The next generation sequencing (NGS) approach used is to resequence the protein coding regions (exome resequencing). For this purpose, exonic DNA from human samples is captured using manufacturer-supplied target fragment enrichment kits, followed by sequencing, for example, using the HiSeq2000 system (Illumina). In addition, tumor mRNA is sequenced to directly quantify gene expression and confirm that mutated genes are expressed in patient tumors. Reading received

- 55 045010 resulting in millions of sequences carried out by software algorithms. The resulting list contains mutations and gene expression. Tumor-specific somatic mutations are identified by comparison with germline variants from PBMCs and prioritized. The de novo identified peptides can then be tested for immunogenicity as described above for the repository, and candidate TUMAP peptides having suitable immunogenicity are selected for inclusion in the vaccine.

In a particular embodiment of the invention, the peptides included in the vaccine are identified by: (a) identifying tumor associated peptides (TUMAP) presented by an individual patient's tumor sample by the methods described above; (b) comparing the peptides identified in step (a) with the peptide repository as described above that have previously been screened for immunogenicity and over-presentation in tumors compared to matched normal tissues; (c) selecting at least one peptide from the repository that correlates with a tumor-associated peptide identified in the patient; and (d) optionally, selecting at least one peptide identified de novo in step (a) with confirmation of its immunogenicity.

In a particular embodiment of the invention, the peptides included in the vaccine are identified by: (a) identifying tumor associated peptides (TUMAP) presented by an individual patient's tumor sample; and (b) selecting at least one peptide identified de novo in step (a) and confirming its immunogenicity.

Once the peptides for the personalized peptide vaccine are selected, the vaccine is manufactured. The vaccine is preferably a liquid dosage form consisting of individual peptides dissolved in DMSO at a concentration of 20-40%, preferably about 30-35%, such as about 33% DMSO.

Each peptide included in the product is dissolved in DMSO. The concentration of individual peptide solutions should be selected depending on the number of peptides intended to be included in the product. Solutions of individual peptides in DMSO are mixed in equal proportions to produce a solution containing all peptides to be included in the product at a concentration of ~2.5 mg/mL per peptide. The mixed solution is then diluted 1:3 with water for injection to achieve a concentration of 0.826 mg/mL per peptide in 33% DMSO. The diluted solution is filtered through a sterile filter with a pore size of 0.22 μm. The final bulk solution is obtained.

The final bulk solution is filled into vials and stored at -20°C until use. One vial contains 700 µl of solution containing 0.578 mg of each peptide. Of this, 500 μL (approx. 400 μg per peptide) will be administered via intradermal injection.

In addition, the peptides of the present invention are useful not only for the treatment of cancer, but also as diagnostic agents. Since the peptides were obtained from lung cancer cells (including NSCLC and SCLC), and since these peptides were determined to be not present or present in small amounts in normal tissues, these peptides can be used to diagnose the presence of cancer.

The presence of the reported peptides on tissue biopsies and blood samples may assist the pathologist in making a diagnosis of cancer. Detection of specific peptides using antibodies, mass spectrometry or other methods known in the art can provide the pathologist with evidence that a tissue sample is malignant or inflamed or affected by disease in general, or can be used as a biomarker for lung cancer (including NSCLC and MRL). The presence of groups of peptides may allow classification or subclassification of affected tissues.

Detection of peptides in diseased tissue samples may allow decisions about the benefit of therapies targeting the immune system, particularly if T cells are known or expected to be involved in the mechanism of action. Lack of MHC expression is a well-described mechanism by which infected or malignant cells evade immune surveillance. Thus, the presence of peptides indicates that this mechanism is not used by the cells analyzed.

The peptides of the present invention can be used in the analysis of lymphocyte responses to the action of these peptides, such as T cell responses or antibody responses to the peptide or peptide complexed with MHC molecules. These lymphocyte responses can be used as prognostic markers to decide on further steps of therapy. These responses can also be used as surrogate response markers in immunotherapeutic approaches aimed at inducing lymphocyte responses through various means, for example, protein vaccination, nucleic acids, autologous materials, adoptive transfer of lymphocytes. In gene therapy settings, lymphocyte responses to peptides can be analyzed to assess side effects. Monitoring lymphocyte responses can also be a valuable tool for post-transplant follow-up examinations, for example, to detect host-versus-graft and graft-versus-host reactions.

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The present invention will be described below by way of examples that describe preferred embodiments thereof, with reference to the accompanying figures, without, however, limiting the scope of the invention. In accordance with the purposes of the present invention, all references cited are incorporated herein in their entirety by reference.

Blueprints

In fig. 1E-N shows the over-presentation of different peptides in different cancer tissues (black dots). Top: Median MS signal intensities of technical replicates are plotted as dots for single positive normal tissue samples (gray dots) and tumor samples (black dots) on which the peptide was detected. Tumor and normal samples are grouped by organ of origin, and range (box-whisker) plots show the median, 25th and 75th percentiles (box), and minimum and maximum (whiskers) of normalized signal intensity for multiple samples. Normal organs are arranged according to risk categories (blood cells, blood vessels, brain, liver, lungs: high risk, gray dots; reproductive organs, breast, prostate: low risk, gray dots; all other organs: medium risk level; gray dots). Bottom panel: The relative frequency of peptide detection in each organ is presented as a bar graph. The numbers below the graph indicate the number of samples in which the peptide was detected out of the total number of samples analyzed for each organ. If a peptide was detected on a sample but could not be quantified for technical reasons, the sample was included in this detection frequency representation plot, but no dot was designated at the top of the figure. In fig. 1A-1B show the over-presentation of various peptides in HLA-A*24 cancer tissues compared to a panel of HLA-A*24 normal tissue samples (N = 19 normal samples, N = 94 tumor samples). Tissues (from left to right): normal samples: blood, vessels (blood vessels); head brain (brain); heart; liver; lungs; kidneys; pituitary. Tumor samples: GBM: glioblastoma; GC: stomach cancer; HCC: hepatocellular carcinoma; NSCLC (non-small cell lung cancer - adenocarcinoma); NSCLC others (non-small cell lung cancer); NSCLC (squamous cell non-small cell lung cancer); SCLC: small cell lung cancer. fig. 1A) gene symbol: URB1, peptide: LYQEILAQL (SEQ ID NO: 62), FIG. 1B) gene symbol: CKAP5, peptide: VYPASKMFPFI (SEQ ID NO.: 65). In fig. Figures 1C-1D show the over-presentation of various peptides in HLA-A*02 serotype cancer tissues compared to a panel of HLA-A*02 serotype normal tissue samples (N = 469 normal samples, N = 528 tumor samples). Tissues (from left to right): Normal samples: blood cells; shelter. vessels (blood vessels); head brain (brain); heart; liver; lungs; fat tissue (adipose tissue); adrenal; bile prot. (bile ducts); urinary n. (bladder); BM (bone marrow); esophagus; eye; bile n. (gallbladder); head and neck; bud; thick quiche (colon); LN (lymph node); nerve; arson zhel. (pancreas); parashield (epithelial body); peritoneum; pituitary; pleura; skel. muscle (skeletal muscle); leather; thin quiche (small intestine); spleen; stomach; Shitov. zhel. (thyroid); trachea; ureter; dairy zhel. (breast); ovary; placenta; rep. zhel. (prostate); testis; fork zhel. (thymus); uterus. Tumor samples: AML: acute myeloid leukemia; BC: breast cancer; CCC: cholangiocell carcinoma; CLL: chronic lymphocytic leukemia; CRC: colorectal cancer; GCC: gallbladder cancer; GBM: glioblastoma; GC: stomach cancer; RCG: cancer of the cardia of the stomach and esophagus; HCC: hepatocellular carcinoma; Head and neck cancer; MEL: melanoma; NHL: non-Hodgkin's lymphoma; NSCLC (non-small cell lung cancer adenocarcinoma); NSCLC others (non-small cell lung cancer); NSCLC (squamous cell non-small cell lung cancer); OC: ovarian cancer; ER: esophageal cancer; PC: pancreatic cancer; Prostate cancer: prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; BCM: bladder carcinoma; REM: endometrial and uterine cancer. fig. 1C) gene symbol: BMS1, peptide: VLYDKDAVYV (SEQ ID NO: 129), Fig. 1D) gene symbol: GORASP2, peptide: NLWGGQGLLGV (SEQ ID NO: 130). In fig. Ιέ-If shows over-presentation of various peptides in cancer tissues with the HLA-A*01 serotype compared with a panel of normal tissue samples with the HLA-A*01 serotype (N = 13 normal samples, N = 40 tumor samples). Tissues (from left to right): Normal samples: blood cells; heads brain (brain); heart; liver; lungs. Tumor samples: GBM: glioblastoma; HCC: hepatocellular carcinoma; NSCLC (non-small cell lung cancer - adenocarcinoma); NSCLC others (non-small cell lung cancer); NSCLC (squamous cell non-small cell lung cancer); SCLC: small cell lung cancer. fig. 1E) gene symbol: ZNF439, peptide: LLDISQKNLY (SEQ ID NO: 154), FIG. 1F) gene symbol: MMP12, peptide: SADDIRGIQSLY (SEQ ID NO: 174). In fig. Figure 1G-1H shows the over-presentation of various peptides in cancer tissues with the HLA-A*03 serotype compared with a panel of normal tissue samples with the HLA-A*03 serotype (N = 12 normal samples, N = 28 tumor samples). Tissues (from left to right): Normal samples: blood cells; shelter. vessels (blood vessels); heads brain (brain); heart; liver; lungs. Tumor samples: GBM: glioblastoma; GC: stomach cancer; NHL: non-Hodgkin's lymphoma; NSCLC (non-small cell lung cancer - adenocarcinoma); NSCLC others (non-small cell lung cancer); NSCLCflat (flat

- 57 045010 exact non-small cell lung cancer); SCLC: small cell lung cancer. Fig. 1G) gene symbols: KRT81, KRT121P, KRT83, KRT85, KRT86, peptide: KLAELEGALQK (SEQ ID NO: 202), Fig. 1H) gene symbol: NDC80, peptide: SINKPTSER (SEQ ID NO: 457). In fig. Figures 1I-1J show the over-presentation of various peptides in HLA-B*07 serotype cancer tissues compared with a panel of HLA-B*07 serotype normal tissue samples (N = 13 normal samples, N = 36 tumor samples). Tissues (from left to right): Normal samples: blood cells; blood, vessels (blood vessels); heads brain (brain); heart; liver; lungs. Tumor samples: GBM: glioblastoma; NSCLC (non-small cell lung cancer - adenocarcinoma); NSCLC others (non-small cell lung cancer); NSCLC (squamous cell non-small cell lung cancer); OC: ovarian cancer; SCLC: small cell lung cancer. Fig. 1I) gene symbol: CTHRC1, peptide: SPQRLRGLL (SEQ ID NO: 291); fig. 1J) gene symbol: MANEA, peptide: RPHKPGLYL (SEQ ID NO: 475). In fig. 1K-1L shows the over-presentation of various peptides in HLA-B*08 serotype cancer tissues compared with a panel of HLA-B*08 serotype normal tissue samples (N = 1 normal sample, N = 22 tumor samples). Tissues (from left to right): Normal samples: lungs. Tumor samples: GBM: glioblastoma; NSCLC (non-small cell lung cancer - adenocarcinoma); NSCLC others (non-small cell lung cancer); NSCLC (squamous cell non-small cell lung cancer); SCLC: small cell lung cancer. Fig. 1K) gene symbol: VPS13B, peptide: DIYQRALNL (SEQ ID NO.: 315), Fig. 1L) gene symbol: ARID4A, peptide: LVKVKVLL (SEQ ID NO: 317). In fig. 1M-1N shows the over-presentation of various peptides in HLA-B*44 serotype cancer tissues compared to a panel of HLA-B*44 serotype normal tissue samples (N = 15 normal samples, N = 25 tumor samples). Tissues (from left to right): Normal samples: smut. brain (brain); heart; liver; lungs. Tumor samples: GBM: glioblastoma; NSCLC (non-small cell lung cancer - adenocarcinoma); NSCLC others (non-small cell lung cancer); NSCLC (squamous cell non-small cell lung cancer); SCLC: small cell lung cancer. Fig. 1M) gene symbol: NUP155, peptide: SEKGVIQVY (SEQ ID NO: 362), FIG. IN) gene symbol: CLSPN, peptide: SEIGKAVGF (SEQ ID NO: 489).

In fig. 2A-2N show examples of exon expression profiles of parent genes of the present invention that are overexpressed in various cancer samples. Tumor (black dots) and normal (gray dots) samples are grouped by organ of origin, and range (whisker box) plots show the median, 25th and 75th percentiles (box), and minimum and maximum (whisker) RPKM values. Normal organs are arranged according to risk categories. FPKM = fragments per kilobase per million mapped reads. Normal samples: blood cells; shelter. vessels: blood vessels; brain; heart; liver; lung; adipose tissue; adrenal; bile prot.: bile ducts; urinary n.: bladder; BM: bone marrow; cartilage. tissue: cartilage tissue; esophagus; eye; bile n.: gallbladder; head and neck; bud; thick intestine: large intestine; LN: lymph node; nerve; arson gland: pancreas; parathyroid: parathyroid gland; peritoneum; pituitary; pleura; skel. muscle: skeletal muscle; leather; thin intestine: small intestine; spleen; stomach; Shitov. gland: thyroid gland; trachea; ureter; dairy gland: mammary gland; ovary; placenta; rep. gland: prostate gland; testis; fork gland: thymus gland; uterus. Tumor samples: AML: acute myeloid leukemia; BC: breast cancer; CCC: cholangiocell carcinoma; CLL: chronic lymphocytic leukemia; CRC: colorectal cancer; GCC: gallbladder cancer; GBM: glioblastoma; GC: stomach cancer; HCC: hepatocellular carcinoma; Head and neck cancer; MEL: melanoma; NHL: non-Hodgkin's lymphoma; NSCLC: non-small cell lung cancer - adenocarcinoma; NSCLCothers: non-small cell lung cancer; NSCLC squamous: squamous cell non-small cell lung cancer; OC: ovarian cancer; ER: esophageal cancer; PC: pancreatic cancer; Prostate cancer: prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; BCM: bladder carcinoma; REM: endometrial cancer of the uterus. Fig. 2A) gene symbol: ADAMTS12, peptide: QYDPTPLTW (SEQ ID No: 1), FIG. 2B) gene symbol: MMP12, peptide: VWSNVTPLKF (SEQ ID No: 2), Fig. 2C) gene symbol: MMP12, peptide: YVDINTFRL (SEQ ID No: 84), FIG. 2D) gene symbol: KIF26B, peptide: TLYPYQISQL (SEQ ID No: 87), FIG. 2E) gene symbol: CT83, peptide: NTDNNLAVY (SEQ ID No: 164), FIG. 2F) gene symbol: LAMA1, peptide: VSDSECLSRY (SEQ ID No: 189), FIG. 2G) gene symbol: KIF26B, peptide: KVKDTPGLGK (SEQ ID No: 203), FIG. 2H) gene symbol: SP6, peptide: SLDGAARPK (SEQ ID No: 205), FIG. 2I) gene symbol: PRAME, peptide: SPSVSQLSVL (SEQ ID No: 220), FIG. 2J) gene symbol: MMP1, peptide: NPFYPEVEL (SEQ ID No: 222), FIG. 2K) gene symbol: NLRP2, peptide: FNKRKPLSL (SEQ ID No: 298), FIG. 2L) gene symbol: KIF26B, peptide: VASPKHCVL (SEQ ID No: 300), FIG. 2M) gene symbol: MAGEA3, MAGEA6, peptide: MEVDPIGHVYIF (SEQ ID No: 320), FIG. 2N) gene symbol: MMP12, peptide: QEMQHFLGL (SEQ ID No: 326).

In fig. Figure 3 shows typical results of in vitro peptide-specific responses of CD8+ T cells from a healthy HLA-A*02+ donor. CD8+ T cells were primed using artificial APCs stimulated with monoclonal antibodies to CD28 and HLA-A*02 in complex with a peptide with the sequence Seq ID No. 520 (KIQEMQHFL, Seq ID NO: 520) (A, left section). After three cycles of stimulation, cells reacting with the peptide were detected using double staining with A*02/Seq ID 520 multimers (A). The right section (B) represents the control color

- 58 045010 sewing of cells stimulated with irrelevant peptide and A*02 complexes. CD8+ lymphocytes were isolated from individual viable cells by gating. Boole gates helped eliminate false-positive responses detected by multimers specific for different peptides. The frequency of detection of specific multimer-positive cells among CD8+ lymphocytes is indicated.

In fig. Figure 4 shows typical results of in vitro peptide-specific responses of CD8+ T cells from a healthy HLA-A*24+ donor. CD8+ T cells were primed using artificial APCs stimulated with monoclonal antibodies to CD28 and HLA-A*24 in complex with a peptide with the sequence Seq ID No 504 (A, left section). After three cycles of stimulation, detection of cells reacting with the peptide was done using double staining with A*24/Seq ID No. 504 multimers (VYEKNGYIYF, Seq ID NO: 504) (A). The right section (B) is a control staining of cells stimulated with irrelevant peptide and A*24 complexes. CD8+ lymphocytes were isolated from individual viable cells by gating. Boole gates helped eliminate false-positive responses detected by multimers specific for different peptides. The frequency of detection of specific multimer-positive cells among CD8+ lymphocytes is indicated.

In fig. Figure 5 shows typical results of in vitro responses of peptide-specific CD8+ T cells from a healthy HLA-A*01+ donor. CD8+ T cells were primed using artificial APCs stimulated with monoclonal antibodies to CD28 and HLA-A*01 in complex with a peptide with the sequence SEQ ID No. 153 (KLDRSVFTAY, Seq ID No. 153) (A, left section) and with the peptide having the sequence SEQ ID No. 173 (RTEFNLNQY, Seq ID No. 173) (B, left section), respectively. After three cycles of stimulation, detection of cells reacting with the peptide was done using double staining of multimers A*01/Seq ID No. 153 (A) or A*01/Seq ID No. 173. The right sections (A and B) represent control staining of cells , stimulated by irrelevant complexes of the peptide and A*01. CD8+ lymphocytes were isolated from individual viable cells by gating. Boole gates helped eliminate false-positive responses detected by multimers specific for different peptides.

The frequency of detection of specific multimer-positive cells among CD8+ lymphocytes is indicated.

In fig. Figure 6 shows typical results of in vitro peptide-specific responses of CD8+ T cells from a healthy HLA-A*02+ donor. CD8+ T cells were primed using artificial APCs stimulated with monoclonal antibodies to CD28 and HLA-A*02 in complex with a peptide with the sequence SEQ ID No. 89 (ILSTTMVTV, Seq ID No. 89) (A, left section) and with a peptide having the sequence SEQ ID No. 88 (VQMVITEAQKV, Seq ID No. 88) (B, left section), respectively. After three cycles of stimulation, cells reacting with the peptide were detected by double staining of A*02/Seq ID No. 89 (A) or A*02/Seq ID No. 88 (B) multimers. The right panels (A and B) represent control staining of cells stimulated with irrelevant peptide and A*02 complexes. CD8+ lymphocytes were isolated from individual viable cells by gating. Boole gates helped eliminate false-positive responses detected by multimers specific for different peptides. The frequency of detection of specific multimer-positive cells among CD8+ lymphocytes is indicated.

In fig. Figure 7 shows typical results of in vitro responses of peptide-specific CD8+ T cells from a healthy HLA-A*03+ donor. CD8+ T cells were primed using artificial APCs stimulated with monoclonal antibodies to CD28 and HLA-A*03 in complex with a peptide with the sequence SEQ ID No. 208 (GLASRILDAK, Seq ID No. 208) (A, left section) and with a peptide with the sequence SEQ ID No. 210 (ATSGVPVYK, Seq ID No. 210) (B, left section), respectively. After three cycles of stimulation, detection of cells reacting with the peptide was done using double staining of A*03/Seq ID No. 208 (A) or A*03/Seq ID No. 210 (B) multimers. The right panels (A and B) represent control staining of cells stimulated with irrelevant peptide and A*03 complexes. CD8+ lymphocytes were isolated from individual viable cells by gating. Boole gates helped eliminate false-positive responses detected by multimers specific for different peptides.

The frequency of detection of specific multimer-positive cells among CD8+ lymphocytes is indicated.

In fig. Figure 8 shows typical results of in vitro peptide-specific responses of CD8+ T cells from a healthy HLA-A*24+ donor. CD8+ T cells were primed using artificial APCs stimulated with monoclonal antibodies to CD28 and HLA-A*24 in complex with a peptide with the sequence SEQ ID No. 15 (KYALLLQDL, Seq ID No. 15) (A, left section) and with a peptide with the sequence SEQ ID No. 11 (YYSKSVGFMQW, Seq ID No. 11) (B, left section), respectively. After three cycles of stimulation, detection of cells reacting with the peptide was made using double staining of A*24/Seq ID No. 15 (A) or A*24/Seq ID No. 11 (B) multimers. The right sections (A and B) represent control staining of cells stimulated with irrelevant

- 59 045010 complexes of peptide and A*24. CD8+ lymphocytes were isolated from individual viable cells by gating. Boole gates helped eliminate false-positive responses detected by multimers specific for different peptides. The frequency of detection of specific multimer-positive cells among CD8+ lymphocytes is indicated.

In fig. Figure 9 shows typical results of in vitro responses of peptide-specific CD8+ T cells from a healthy HLA-B*07+ donor. CD8+ T cells were primed using artificial APCs stimulated with monoclonal antibodies to CD28 and HLA-B*07 in complex with a peptide with the sequence SEQ ID No. 225 (LPFDGPGGIL, Seq ID No. 225) (A, left section) and with the peptide of SEQ ID No. 248 (IPNWARQDL, Seq ID No. 248) (B, left section), respectively. After three cycles of stimulation, cells reacting with the peptide were detected by double staining of multimers B*07/Seq ID No. 225 (A) or B*07/Seq ID No. 248 (B). The right sections (A and B) represent control staining of cells stimulated with irrelevant peptide and B*07 complexes. CD8+ lymphocytes were isolated from individual viable cells by gating. Boole gates helped eliminate false-positive responses detected by multimers specific for different peptides.

The frequency of detection of specific multimer-positive cells among CD8+ lymphocytes is indicated.

In fig. Figure 10 shows typical results of in vitro responses of peptide-specific CD8+ T cells from a healthy HLA-B*08+ donor. CD8+ T cells were primed using artificial APCs stimulated with monoclonal antibodies to CD28 and HLA-B*08 in complex with a peptide with the sequence SEQ ID No. 299 (MAQFKEISL, Seq ID No. 299) (A, left section) and with a peptide having the sequence SEQ ID No. 297 (RAQLKLVAL, Seq ID No. 297) (B, left section), respectively. After three cycles of stimulation, cells reacting with the peptide were detected by double staining of B*08/Seq ID No. 299 (A) or B*08/Seq ID No. 297 (B) multimers. The right panels (A and B) represent control staining of cells stimulated with irrelevant peptide and B*08 complexes. CD8+ lymphocytes were isolated from individual viable cells by gating. Boole gates helped eliminate false-positive responses detected by multimers specific for different peptides. The frequency of detection of specific multimer-positive cells among CD8+ lymphocytes is indicated.

In fig. Figure 11 shows typical results of in vitro responses of peptide-specific CD8+ T cells from a healthy HLA-B*44+ donor. CD8+ T cells were primed using artificial APCs stimulated with monoclonal antibodies to CD28 and HLA-B*44 in complex with a peptide with the sequence SEQ ID No. 325 (QEQDVDLVQKY, Seq ID No. 325) (A, left section) and with a peptide with the sequence SEQ ID No. 331 (EDAQGHIW, Seq ID No. 331) (B, left section), respectively. After three cycles of stimulation, peptide-reactive cells were detected by double multimer staining and CD8+ lymphocytes with B*44/Seq ID No. 325 (A) or B*44/Seq ID No. 331 (B) were isolated from individual cells by gating. The right panels (A and B) represent control staining of cells stimulated with irrelevant peptide and B*44 complexes. CD8+ lymphocytes were isolated from individual viable cells by gating. Boole gates helped eliminate false-positive responses detected by multimers specific for different peptides. The frequency of detection of specific multimer-positive cells among CD8+ lymphocytes is indicated.

Examples

Example 1. Identification and quantification of tumor-associated peptides presented on the cell surface.

Fabric samples.

Patient tumor tissues were obtained from: Asterand (Detroit, MI, USA and Royston, Hertfordshire, UK); Bio-Options Inc. (Bree, California, USA); Geneticist Inc. (Glendale, California, USA); University Hospital of Heidelberg (Heidelberg, Germany); ProteoGenex Inc. (Culver City, California, USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Munich (Munich, Germany). Normal tissues were obtained from Asterand (Detroit, MI, USA and Royston, Hertfordshire, UK); Bio-Options Inc. (Bree, California, USA); BioServe (Beltsville, MD, USA); Capital BioScience Inc. (Rockville, Maryland, USA); Center for Clinical Transfusiology in Tübingen (Tübingen, Germany); Geneticist Inc. (Glendale, California, USA); Kyoto Prefectural University of Medicine (KPUM) (Kyoto, Japan); Osaka City University (OCU) (Osaka, Japan); ProteoGenex Inc. (Culver City, California, USA), Tissue Solutions Ltd (Glasgow, UK), University Hospital of Geneva (Geneva, Switzerland), University Hospital of Heidelberg (Heidelberg, Germany), University Hospital of Tübingen (Tübingen, Germany ); University Hospital Munich (Munich, Germany). Written informed consent was obtained from all patients before surgery or autopsy. Immediately after removal, tissues were shock-frozen and stored at −70°C or lower until TUMAP peptides were isolated.

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Isolation of HLA peptides from tissue samples.

Pools of HLA peptides from shock-frozen tissue samples were obtained by immunoprecipitation from solid tissues according to a slightly modified protocol (Falk et al., 1991; Seeger et al., 1999) using the HLA-A*02-specific antibody BB7.2 , HLA-A, -B, -C-specific antibody W6/32, HLA-DR-specific antibody L243 and HLA-DP-specific antibody B7/21, CNBr-activated Sepharose, acid treatment and ultrafiltration.

Mass spectrometric analysis.

The resulting pools of peptide-HLA complexes were separated according to their hydrophobicity by reverse phase chromatography (nanoAcquity UPLC system, Waters), and the eluted peptides were analyzed on LTQ-velos and -fusion hybrid mass spectrometers (ThermoElectron) equipped with an ESI source. Peptide pools were applied directly to an analytical fused silica microcapillary column (75 μm i.d. x 250 mm) with 1.7 μm C18 reverse phase sorbent (Waters) using a flow rate of 400 nL per minute. The peptides were then separated using a two-step, 180-minute binary gradient from 10 to 33% solvent B at a flow rate of 300 nL per minute. To create the gradient, solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) were used. A gold-plated glass capillary (PicoTip, New Objective) was used to introduce the nano-ESI source. LTQ-Orbitrap mass spectrometers operated in information-dependent mode using the TOP5 strategy. Briefly, the scan cycle started with a full high-mass accuracy scan on the Orbitrap spectrometer (R = 30,000), followed by an MS/MS scan also on the Orbitrap (R = 7500) on 5 particularly abundant precursor ions, with dynamic exclusion of previously selected ions. Tandem mass spectra were interpreted using SEQUEST programs with a fixed false-positive detection rate (q<0.05) and additional manual control. If the identified peptide sequence was uncertain, it was further confirmed by comparing the resulting fragmentation pattern of the natural peptide with the fragmentation pattern of a synthetic control peptide with an identical sequence.

Relative quantitation by LC/MS without isotopic labels was performed by ion counting, i.e. using extraction and LC/MS analysis (Mueller et al., 2007). This method is based on the assumption that the peak area of the LC/MS signal of a peptide correlates with its concentration in the sample. The extracted features were processed using charge state deconvolution analysis and retention time alignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC/MS spectral results were cross-referenced with sequence identification results to combine quantitative data from different samples and tissues into peptide presentation profiles. Quantitative data were normalized using a two-level system according to central tendency to account for variability within technical and biological replicates. In this way, each identified peptide can be associated with quantitative data allowing for relative quantification of samples and tissues. In addition, all quantitative data obtained for candidate peptides were manually checked to ensure inter-data consistency and to verify the accuracy of the automated analysis method. For each peptide, a presentation profile was calculated showing the average level of presentation in the sample as well as the variability of the replicates. The profile compares cancer tissue samples to a background level of normal tissue samples. The presentation profiles of peptides individually or exclusively presented in excess on tumors are shown in FIG. 1.

In table 8 shows the presentation of selected peptides in various cancers and thus the specific relevance of said peptides for the diagnosis and/or treatment of said cancers (e.g., peptide of SEQ ID No. 3 for hepatocellular carcinoma, peptide of SEQ ID No. 11 for melanoma, ovarian cancer and uterine cancer).

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Table 8

Summary of Presentation Data of Selected Tumor-Associated Peptides of the Invention Among Various Species

SEQ ID No. Subsequence Presentation of peptides in cancer diseases 3 YLEKFYGL GKK 6 KYKDYFPVI GKK 9 RILRFPWQL CHALK eleven YYSKSVGFMQW MEL, RYA, REM 13 HYTYILEVF RJ, REM 14 SYSSCYSF RJ 18 DYIGSVEKW RPrZh 19 ILKEDPFLF RY, PKK 21 SYEVRSTF HGK, RY, RPrZh 22 TQPGDWTLF CHALK 23 KFIISDWRF CHALK 24 MYPDLSELLM AML, GBM, RY, SEM 26 KTPTNYYLF RJ 28 YYSIISHTL GKK 31 QYQNVLTLW GBM, RZh, GKK, MEL, NHL, RPrZh, PKK, REM 32 SLPDLTPTF RPrZh 33 KSSVIASLLF GBM 34 MQPRMFFLF OML, GBM, GKK, MEL, SEM 36 KQMEDGHTLF OML, RY 37 QWPWQASLQF RJ 38 KYTNWKAFL OML, GKK 41 VIYFMGAIF GKK, RPrZh, REM 43 IQMDEPMAF OML, MEL, RY 44 AYLSAVGTF OML, RJ, MEL 45 KYFVPPQLF RJ 47 KYADYFLEV GBM, RY, REM 48 VFIDHPVHLKF SEM

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50 SYPELVKMVW AML, RJ, ΓΚK 51 KYALLLQEL AML, CLL, GBM, RJ, GKK, MEL, RYA, RPrZh, PKK, REM 52 KYMKIFHKF RY, REM 53 KYITNLEDL RPrZh 54 LLIKLLQTF AML, GBM, MEL, RY, RPrZh, PKK 55 RWMDQRLVF RJ, GKK, MEL, REM 56 VYMIEPLEL GBM, NHL 57 YPSIIQEF GBM, PKK 58 QFAAPLRGIYF GBM, GKK 59 KYSTTFFMV GBM 60 TYLSIFDQL AML, RJ, GKK, RY 61 NYAENILTL OML, GBM, RJ, MEL 62 LYQEILAQL AML, GBM, RJ, GKK, MEL, RPrZh, PKK 63 VMPSDSFFF MEL, NHL, RY, REM 64 NYAIFDEGHML GBM, RJ 65 VYPASKMFPFI GBM, RJ, GKK, MEL, NHL, RYA, REM 66 IYFRDSSFL OML, RJ, MEL 67 RYPGKFYRV RY 68 IYQQIIQTY RJ, MEL, REM 69 IMPEKFEFW OML, RJ, MEL, NHL, REM 70 PYTNYTFDF RJ, MEL 71 SYMVLAPVF CHALK 72 RYEGILYTI RJ, GKK, NHL, RPrZh 73 SYIGLPLTL RJ, GKK, PKK 74 VYDQYFITL AML, prostate cancer 76 WYGWHFPEL OML, RJ, GKK, PKK, REM 77 AYTLLGHEFV RJ, MEL, RYA 78 TWFPKTPMLF OML, GBM, RJ, GKK, MEL, SEM 79 RYLADLPTL RJ, GKK, RYA, REM 80 YYSPLRDLL CHALK 82 RFLPSPWI AML, RJ, GKK, MEL, RPrZh, REM 83 TYCQNIKEF AML, RY, RP, REM 84 YVDINTFRL GKK 86 FVIDGFDEL KRK, RJ, NHL, RP 87 TLYPYQISQL GKK, RP 90 FLLMHPSI HLL, GKK, PKK 91 FALPGLLHA RJ, GKK, NHL, RP, PKK 92 NLRDLLSEV GKK 93 TLQEKILQV CLL, GBM, NHL 95 ITIGVLARV KRK, RPZh 96 HLVGGLHTV AML, RMZH, KRK, RZH, MEL, RYA, RPrZH

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97 VLALVNSTV CLL 98 LQSSGLTLLL GBM, RY, RPrZh 99 FLKEKVPGI HLL, RJ, MEL, NHL 100 RQYPTPFQL AML, CLL, KRC, GBM, NHL, RY, PCC 101 FIISDWRFVL PlKGSH 102 SLLEQAIAL RJ, GKK, NHL, RY, REM 103 FLYYPDPVL RZhP, GKK, MEL, NHL 105 SLLTHIPTA OML, KRK, PlKGSH, MEL, RYA, RP, PKK, RMP, REM 106 FIIDTTYPAYV KRK, RY, RP 107 LLQGAIESV RMJ, CLL, KRK, PlKGSH, MEL, NHL, RMP, REM 108 MIIALSLY AML, breast cancer 110 LLADFQALL PAC 111 ALCLLLHLL OML, RZhP, GKK, PlKGSH, PKK 113 AVLTGLVEV RMJ, CLL, KRK, RJ, GKK, NHL, RP, PKK, REM 114 ILDERQVLL AML, RMJ, KRK, RZhP, RZh, GKK, PlKGSh, MEL, NHL, RY, RP, RP, RMP, REM 115 MLLETQDALYV PlKGSH 116 VLMEENSKL GBM 117 FLDPNARPLV NHL, RY 118 ALSSVLHSI AML, RMJ, KRK, RZhP, GKK, PlKGSH, MEL, NHL, RYA, RP, RPZh, PKK, RMP 119 RTADITVTV OML, KRK, RMP 120 ALLANLPAV RZhP, RZh, RYA, RZhP 121 ALVDTLTGI PlKGSH, NHL, REM 122 ALLEMFPEITV RMJ, RY, RPrZh 123 LMAFFLAVV HGK, GKK, NHL, RP, PKK 124 SVASVLLYL AML, BC, CLL, GCC, PKGSH, NHL, RY 125 VLQPFLPSI AML, RMJ, HGK, CLL, KRK, GKK, PlKGSh, MEL, NHL, RY, PKK 126 FLSTVTSV HGK, GBM, GKK, PlKGSH, MEL, NHL, RP, RPZh, PKK, RMP, REM 127 GLDGSLVFL AML, CLL, KRK, GBM, GKK, PlKGSH, MEL, NHL, RYA, RP, RMP 128 FLGTTPTL AML, CLL, GBM, PKGSH, NHL, OC, RMP, REM 129 VLYDKDAVYV AML, CLL, KRK, PlKGS, NHL, RY, RMP, REM 130 NLWGGQGLLGV RMZh, RZhP, GBM, RZh, GKK, RY, RPrZh, REM 131 LLKEFVQRV RMZH, KRK, GKK, PlKGSh, RY, RP 132 ALWLVDPLTV CLL, KRK, GBM, GKK 133 MTLPVDAVISV HLL, KRK, GKK, PlKGSH, NHL, RPrZh, REM 134 AAEIGDKSWLY RJ, MEL, NHL, REM 135 ASEDSVLLY GBM, RZh, MEL, NHL, RP, RPrZh,

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136 ATDLVVLDRY GBM, RZh, GKK, MEL, NHL, RP, RPrZh, REM 137 ATSKFMEFY GBM, MEL, RYA, RPrZh, REM 139 ECDMAFHIY MEL, RYA 140 ESDREELNY RJ, RPrZh 141 ESDVGWVY GBM, RJ 142 EVAEPSVLFDLY RJ, MEL, NHL, RY, REM 144 FLDSQNLSAY RJ 145 FVDKPVAY GBM, RJ, MEL 146 GLNTGSALSY RJ, RY 148 GTEFTTILY RJ, NHL, RP, RPrZh 149 GTEFTTVLY RZh, PlKGSh, MEL, RP, RPrZh 150 GTELLSLVY RJ, MEL, NHL, RYA, RPrZh, REM 152 HTDSLHLLI RJ, MEL, RYA 154 LLDISQKNLY GBM, NHL, RPrZh 155 LLDPNPHMY RPrZh 156 LLDSLREQY RJ, NHL, RP 157 LMDRPIFY GBM, RJ, MEL, NHL 159 LSDTSVIQFY GBM, RZh, GKK, MEL, NHL, RPrZh 160 LTEAVLNRY RY 161 LVDDGTHGQY GBM, RJ, MEL 162 LVDNSIRELQY RJ, GKK, MEL, NHL, RY, REM 163 NSDSSLTLREFY GKK, RPrZh 166 NTQITDIGRY CHALK 167 QSDPGTSVLGY GBM 169 RLDTPLYFSY MEL, RYA 170 RSDDTAVYY CLL, RJ, GKK, NHL, RPG, REM 172 RTDSCSSAQAQY CHALK 173 RTEFNLNQY RZhP, RZh, MEL, REM 177 SSDEVNFLVY RJ, CHALK, RYA, REM 178 SSDSSTLPKL RJ, RY, RPrZh 179 STAKSATWTY RPrZh 180 STDPWIQMAY GBM, RJ, MEL 181 TADGKTYYY GBM, GKK, MEL, RPrZh 182 TDYHVRVY GBM, RPrZh 184 TSAHPEDSSFY RJ, NHL, RPrZh 186 TTDIIEKY RJ, MEL, 187 VADLHLY RJ, MEL, RYA, RPrZh, PKK 190 VTDGINPLIDRY CHALK 191 VTDGSLYEGVAY RJ, MEL, REM 192 VTEESFDSKFY RJ 193 VTEFSLNTY RZhP, RZh, MEL, RYA, RP, REM 196 WMFFVINY SEM

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197 YADTVRPEFY RY 198 YLDPVQRDLY GBM, RJ, GKK, MEL, NHL, RYA, REM 202 KLAELEGALQK MEL, RYA, REM 204 AVFDKFIRY GBM 207 RSFNGLLTMY MEL, RYA 210 ATSGVPVYK SEM 211 TVNPVAIHK GBM, NHL, RY, RPrZh, REM 212 KAYEQVMHY SEM 213 LNINMTSPMGTK GBM, RJ, MEL, NHL, RPZh 214 RTMSEAALVRK RJ, NHL, RY, RPrZh, REM 215 MMFSGPQILKL CHALK 216 KLYAWELAF AML, GBM, MEL, RY, RP 217 RILNQILYY AML, GBM, RJ, GKK, MEL, NHL, RPrZh, REM 218 KTLVAELLILK AML, NHL, REM 219 RLRSSLVFK SEM 220 SPSVSQLSVL SEM 235 MPLKHYLLL MEL, NHL, RY, REM 237 RPAATAVISL GBM, NHL, RY 244 FPYVRDFVM RZhP, MEL, NHL, RYA, REM 247 RALLARLLLL NHL 251 VPRSSGQTV RMZH, GBM, REM 255 MPLLENLYL RY, REM 256 SPRVPSIEL NHL 259 RPPAAGLRGISL GBM 260 YPQHPGLNA RMJ, GBM, RG, NHL 262 SAYPQRLEI RJ 263 HPAPYGDLL SEM 271 MPLPWSLALP CHALK 273 MPLLWLRGF SEM 274 TPYQEHVAL RY 275 APHPPLSVV AML, BC, MEL 276 LPRAGGAFL NHL, RY, PKK, REM 277 MPLFEPRVF RY, REM 278 HPMIDINGIIVF SEM 280 VPISEEGTPVL MEL, RYA, RPrZh, REM 281 RPRAPVTPA GBM 282 MPQIETRVIL SEM 283 RPHSLSSEL OML, NHL 284 FPVTSIFHTF MEL, RYA, REM 285 FPSFLTNSL AML 286 VPTLRSEL RY 288 FPQKFIDLL SEM

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289 VPENHSVAL SEM 290 APYRPPDISL RMJ 292 SPQRLRGLLL NHL 293 RPRSALPRLLLP NHL, REM 295 KPEGTRIAV NHL, REM 296 MPMQDIKM SEM 300 VASPKHCVL RY 301 YMHKLLLVL AML, NHL, RY, RP, REM 305 ALKLRVAVL NHL 306 ILKVKVGL MEL, RYA 308 MLKQKVEEL RP 311 EIRIRWQM MEL, NHL, RY, RPrZh 313 ELKKKEYEEL NHL 314 AIISRLVAL NHL, RP, RMP 316 VIKEKALTL NHL, RY, RPrZh, pkk 318 EAAIRSVEL GBM, MEL, NHL, RY 321 AEMLESVIKNY NHL 322 KEVDPAGHSY NHL 323 SEFMQVIF NHL 328 FEYDFLLQRI SEM 330 KEGDLGGKQW NHL 335 KELEATKQY MEL, NHL 337 TENRYCVQL gkk 342 HEFSSPSHL NHL 343 TEFTTVLY GBM, NHL, RPrZh 345 IEFIHPQAF GBM, RJ, NHL, RPrZh 347 ALNPYQYQY SEM 348 AEIQGNINHV SEM 351 EEVNYINTF OML, MEL, NHL, RY 354 TEDPTILRI RJ, GKK, RYA, RPrZh, REM 356 EEGRVYLF GBM, RPrZh 357 RELENCFQIQ SEM 359 DELFSIALY NHL 363 AELDKLTSV GBM, SEM 366 AENLFRAF GBM, NHL, RY 367 GEVHPSEMI SEM 368 GEFPVVRVQV AML, RJ, RY, REM 370 YEDLSQKY GBM, NHL, RY 371 GELALKKKI SEM 372 TEGIIMKDF RY, RPrZh, PKK, REM 373 MEMQKSPVF NHL, RY 374 DEVNFLVY HGK, RZhP, GBM, NHL, RY, RPrZh

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375 VYSDLHAFYY GBM, RZh, GKK, MEL, RPrZh 376 KYVKDFHKF AML, RY, RP, REM 377 VYVGAVNRI RJ, GKK, RPrZh 378 KFLGPAEHLTF RY 379 NYIVPDKQIF GBM, RZh, GKK, MEL, RYA, RPrZh 380 VFQEKHHVI RPrZh 381 TYSKKHFRI MEL, RYA 382 IYHSHHPTL OML, MEL, NHL, RY, REM 383 RYKQDVERF AML, MEL, RY, RPrZh, REM 384 KYVKVFDKF AML, REM 385 MYINEVERL GBM, MEL, RYA, RPrZh, REM 386 VYNDHSIYVW AML, GBM, GKK, MEL, NHL, RPrZh, REM 387 RWLPQKNAAQF OML, RJ, GKK, MEL, RYA, PKK, REM 388 FSIPEGALVAV AML, HGK, CLL, KRK, RZhP, RJ, GKK, PlKGSH, MEL, NHL, RY, RP, RPrZh, PKK, RMP, REM 389 TLMEQPLTTL AML, RMJ, KRK, GKK, PlKGSH, NHL, RY, RPrZh, REM 390 HIMPTVHTV Breast cancer, CLL, GBM, GKK, PlKGS, MEL, NHL, RY, RP, RMP, REM 391 SLIDMRGIETV Breast cancer, CLL, GBM, GCC, PlCGSH, OC, RMP 392 SLFKDQMEL AML, RMJ, CLL, KRK, GBM, GKK, MEL, NHL, OC, RPZH, PKK, RMP, REM 393 ILLPYLQTL AML, BC, CLL, KRK, GBM, GKK, PlKGS, NHL, RY 394 ASEAEMRLFY GBM, RJ, MEL, NHL 395 ASEASRLAHY GBM, RJ, NHL, RPrZh, REM 396 ASEFGNHYLY GBM, RZH, MEL, REM 397 ASEITSKGASLY GBM, RZh, GKK, MEL, RYA, RPrZh 398 ASEQQALHTVQY GBM, RZh, MEL, NHL, RPrZh, REM 399 ATDIPCLLY CHALK 400 ATDISRQNEY GBM, RJ, NHL, RY 401 DDSDESYMEKSLY RJ 402 DTDSQRLAY GBM, RJ, MEL 403 ELDSKVEVLTY GBM, RZh, MEL, RYA, RPrZh 404 ETARKFLYY GBM 405 ETEEGIYWRY RJ 406 ETEQTKFWDY GBM, RZH, MEL, RYA, PKK, REM 407 FSDNDKLYLY GBM, RZh, MEL, NHL, RPrZh, REM 408 FTEQWTDGY GBM, RZh, RYa, RP, RPrZh, 409 FVDPLVTNY GBM, RZh, GKK, MEL, NHL, RYA, RP, RPrZh, PKK 410 GSDHQSPSSSSSY GBM, RJ, MEL, RYA, RPrZh, 411 GTVYEDLRY GBM, RJ, GKK, MEL, NHL, RY, RP 412 ILDEVIMGY GBM, RJ, MEL, NHL, RY, RP

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413 ISDRYYTALY GBM, RZh, RPrzh, REM 414 KTDESLTKY RJ, NHL, RPrZh 415 LLDPRSYHTY RJ, NHL 416 LLDTAQKNLY GBM, NHL, RPrZh 417 LLEDKHFQSY GBM, RZh, GKK, MEL, NHL, RYA, RPrZh, REM 418 LSDPSGPKSY GBM, GKK, RPrZh 419 LSELKPMSY GBM, RZh, GKK, MEL, RP, RPrZh, PKK, REM 420 LTEDKETLQY RJ, GKK, MEL, NHL, RPrZh 421 LTELLERAAFY RJ, MEL, NHL, REM 422 MIDVTKSYY GBM, RZh, MEL, NHL, RYA, RP, RPrZh, REM 423 NLDAVHDITVAY RJ, MEL, RPrZh, REM 424 NLDEEKQLLY MEL, RPrZh 425 NLDIIQQEY GBM, RZh, GKK, MEL, NHL, RYA, RP, RPrZh, REM 426 NLDQATRVAY NHL 427 NSDEQKITEMVY RJ 428 NSELSCQLY GBM, RZh, MEL, PKK 429 NTEDSSMSGYLY RJ, MEL, 430 NTEGLHHLY GBM, GKK, MEL, RPrZh 431 NTSDMMGRMSY RJ, MEL, NHL, RY, 432 NVDPVQHTY GBM, RZh, GKK, PlKGSH, MEL, NHL, RYA, RP, RPrZh, PKK, REM 433 QIDTGENLY RJ, MEL 434 QTDCAPNNGY GBM, RZH, MEL, RPrZH 435 QTDDTTWRTEY GBM, RJ, MEL, NHL, RPrZh 436 QTETGTPYMLY GBM, RZh, MEL, NHL, RYA, RP, RPrZh, REM 437 STDGKHWWEY RJ, MEL, NHL, RPrZh 438 STDNFNCKY RJ, GKK, MEL 439 TLDAGKFQIY RJ, GKK, MEL, NHL, RPrZh, REM 440 TLDENPGVRY GBM, RZh, GKK, MEL, NHL, RPrZh, REM 441 TLDSALNAASYY GBM, RJ, MEL, NHL, RPrZh, PKK 442 TSDFSRFTNY GBM 443 TTDFPSESSFEY RJ, MEL, NHL, RYA, RPrZh, REM 444 TTDTVIRSY RJ, MEL, REM 445 VLDQGKITEY GBM, GKK 446 VTAQVVGTERY RJ, MEL 447 VVDEDHELIY GBM 448 YLDIPNPRY GBM, MEL, PKK 449 YLDRGTGNVSFY GBM, RZh, GKK, MEL, RPrZh, PKK 450 YSDDGQKWTVY CHALK 451 YSDSLVQKGY GBM, RZh, GKK, RYA, RPrZh, REM 452 YVDAVLGKGHQY GBM, RZh, MEL, NHL, RYA, RPrZh, REM

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453 AINTSIKNK RPrZh 454 KVYTPSISK GBM, GKK, MEL, REM 455 RIADIFVKK RJ, MEL, NHL, RY, REM 456 SMFTAILKK OML, MEL, NHL, RY, REM 457 SINKPTSER GBM 458 GIADFVLKY AML, RMJ, GBM, MEL, NHL, REM 461 RPILIIVTL NHL 464 YPRPGTPAA AML, RJ, MEL, NHL, RY, PKK 465 VPRPIFSQL NHL 468 SPMYGQAGL PAC 469 YPENGWQM OML, RY 470 SPNSYFRVL PAC 471 KPRPDVTNEL NHL 472 NPRATDAQL AML 473 LPRALLSSL NHL, RY 474 LPRLLPAL OML, NHL 476 AEEEEIMKKI NHL 477 QENSYQSRL RY 479 AEIQPQTQV SEM 480 GEVSGLTKDF MEL, NHL, RY 481 RELQHEHSL RY 482 TEREWADEW OML, MEL, NHL, RY, PKK 483 EENDQSTHKW MEL, NHL, RY, PKK, REM 484 AEVGFVRFF OML, MEL, NHL, RY 485 SEIEDSTKQVF MEL, NHL, RY 486 SEDDPILQI NHL, RY, REM 487 AEDQLHHSF OML, NHL 488 TEFPIIKMY AML, MEL, NHL, RY, RP

AML = acute myeloid leukemia, BC = breast cancer, CGC = bile duct cancer, GBM = brain cancer, CLL = chronic lymphocytic leukemia, CRC = colorectal carcinoma, esophageal cancer, gall bladder adenocarcinoma, gastric cancer , HCH = head and neck squamous cell carcinoma, HCC = hepatocellular carcinoma, MEL = melanoma, NHL = non-Hodgkin lymphoma, OC = ovarian cancer, PC = pancreatic cancer, PCa = prostate cancer and benign prostatic hyperplasia, RCC = renal cell carcinoma, BCC = bladder cancer, REM = uterine cancer.

Example 2. Determination of the expression profile of genes encoding peptides according to the invention.

Excessive presentation or specific presentation of a peptide on tumor cells compared to normal cells is sufficient for its utility in immunotherapy, and some peptides are tumor specific despite the presence of their parent proteins also in normal tissues. However, identifying mRNA expression profiles adds an additional layer of safety when selecting peptide targets for immunotherapy. Particularly for therapeutics with high safety risks, such as affinity-matured TCRs, the ideal target peptide will be derived from a protein that is unique to the tumor and not found on normal tissues.

Sources and preparation of RNA.

Surgically removed tissue specimens were provided by the various organizations listed above (see Example 1) after obtaining a written informed consent form from each patient. Tumor tissue specimens were snap-frozen after surgery and subsequently homogenized using a mortar and pestle in liquid nitrogen. Total RNA was prepared from these samples using TRI reagent (Ambion, Darmstadt, Germany) followed by purification on RNeasy (QIAGEN, Hilden, Germany); both methods were carried out according to the manufacturers' instructions.

Total RNA from healthy human tissues for RNA sequencing (RNASeq) experiments was obtained from: Asterand Companies (Detroit, MI, USA and Royston, Hertfordshire, UK); Bio-Options Inc. (Bree, California, USA); BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, MD, USA); Capital BioScience Inc. (Rockville, Maryland, USA); Geneticist Inc. (Glendale, California, USA); University Hospital Heidelberg (Thoracic Clinic, Heidelberg, Germany); Istituto Nazionale Tumori Pascale (Naples, Italy); ProteoGenex Inc. (Culver City, California, USA).

Total tumor tissue RNA for RNASeq experiments was obtained from: Asterand (Detroit, MI, USA and Royston, Hertfordshire, UK); Geneticist Inc. (Glendale,

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California, USA); ProteoGenex Inc. (Culver City, California, USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Bonn (Bonn, Germany); University Hospital Tübingen (Tübingen, Germany) The quality and quantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip Kit (Agilent).

RNA sequencing experiments.

Analysis of gene expression in RNA samples of tumor and normal tissue was carried out using next-generation sequencing (RNAseq) by the CeGaT laboratory (Tübingen, Germany). Briefly, sequencing libraries were prepared using the Illumina HiSeq v4 reagent kit according to the manufacturer's protocol (Illumina Inc., San Diego, CA, USA), which includes RNA fragmentation, cDNA synthesis, and addition of sequencing adapters. Libraries obtained from multiple samples were mixed in an equimolar ratio and sequenced on an Illumina HiSeq 2500 system according to the manufacturer's instructions, generating 50-bp single-end reads. The processed reads are mapped to the human genome (GRCh38) using STAR software. Expression data are reported at the transcript level as RPKM (reads per kilobase per million mapped reads using Cufflinks software) and at the exon level (total reads per kilobase using Bedtools software), based on identifications by ensembl sequence data bank (Ensembl77). To obtain RPKM values, exon reads are normalized by exon length and alignment size.

Representative expression profiles of the parent genes of the present invention that are highly overexpressed or exclusively expressed in lung cancer cells (including NSCLC and SCLC) are presented in FIG. 2. Expression rates of other individual genes are shown in Table. 9.

Table 9

Expression indicators

SEQ ID No Subsequence Gene expression NMRLadeno NSCLCflat NSCLCothers MRL 1 QYDPTPLTW +++ +++ + + 2 VWSNVTPLKF +++ +++ + +++ 3 YLEKFYGL +++ +++ +++ 4 SYEKVINYL +++ +++ 5 RYMKKDYLI +++ 6 KYKDYFPVI + +++ ++ 7 VQQWSVAVF +++ 8 PFLPPAACFF +++ 9 RILRFPWQL +++ ++ 10 VWSDVTPLNF ++ +++ eleven YYSKSVGFMQW ++ 12 STIRGELFFF ++ ++ 13 HYTYILEVF ++ ++ 14 SYSSCYSF ++ 15 KYALLLQDL + ++ 16 TYNPDFSSL ++ 17 YYADKKTFIVL + ++ + 18 DYIGSVEKW + + 19 ILKEDPFLF + 20 EFTTVLYNF + 21 SYEVRSTF + + + 22 TQPGDWTLF + 23 KFIISDWRF +

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24 MYPDLSELLM + + 25 SYNGYVFYL + + 26 KTPTNYYLF + 27 NYTLYPITF + 28 YYSIISHTL + + 29 VYPLLSRLYW + + thirty QYLPGWTVLF + 31 QYQNVLTLW + 32 SLPDLTPTF + + 33 KSSVIASLLF + 34 MQPRMFFLF + + 35 KYLEESVWL + 36 KQMEDGHTLF + 37 QWPWQASLQF + 38 KYTNWKAFL + 39 LIFMLANVF + 40 QYEPPSAPSTTF + 41 VIYFMGAIF + 42 TLPNTIYRF + + 43 IQMDEPMAF + 44 AYLSAVGTF + 45 KYFVPPQLF + 46 AFPVTSIFHTF + + 47 KYADYFLEV + 48 VFIDHPVHLKF + 49 LYISEVRNI + 50 SYPELVKMVW + 51 KYALLLQEL + 52 KYMKIFHKF + 53 KYITNLEDL + 54 LLIKLLQTF + 55 RWMDQRLVF + 56 VYMIEPLEL + 57 YPSIIQEF + 84 YVDINTFRL +++ +++ + +++ 85 YIDEFQSLV ++ 86 FVIDGFDEL ++ ++ ++ 87 TLYPYQISQL ++ ++ + ++ 88 VQMVITEAQKV ++ ++ 89 ILSTTMVTV ++ ++ + + 90 FLLMHPSI ++

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91 FALPGLLHA ++ ++ 92 NLRDLLSEV ++ ++ + + 93 TLQEKILQV ++ 94 VLPDIETLIGV ++ ++ 95 ITIGVLARV ++ 96 HLVGGLHTV + 97 VLALVNSTV + 98 LQSSGLTLLL + + + 99 FLKEKVPGI + 100 RQYPTPFQL + + 101 FIISDWRFVL + 102 SLLEQAIAL + 103 FLYYPDPVL + 104 GMLDIFWGV + 105 SLLTHIPTA + 106 FIIDTTYPAYV + 107 LLQGAIESV + 108 MIIALSLY + + 109 LLLGSIGLLGV + 110 LLADFQALL + 111 ALCLLLHLL + 112 SVSDGIHSV + 113 AVLTGLVEV + 114 ILDERQVLL + + 115 MLLETQDALYV + + 116 VLMEENSKL + 117 FLDPNARPLV + 118 ALSSVLHSI + 119 RTADITVTV + + 120 ALLANLPAV + + 121 ALVDTLTGI + 122 ALLEMFPEITV + 123 LMAFFLAVV + 124 SVASVLLYL + 138 DSDSCHFNY ++ 139 ECDMAFHIY + 140 ESDREELNY + 143 FIDYPKKEDY ++ + 146 GLNTGSALSY ++ + 147 GSSDSSTLPKL + 148 GTEFTTILY +

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149 GTEFTTVLY + 150 GTELLSLVY + 153 KLDRSVFTAY ++ 155 LLDPNPHMY + ++ 158 LSDLLKQGY ++ + 160 LTEAVLNRY ++ ++ + ++ 163 NSDSSLTLREFY + + 164 NTDNLAVY +++ +++ + 165 NTDPTAPPY + + 166 NTQITDIGRY + + 167 QSDPGTSVLGY + 168 QTDHPQPILDRY + ++ 171 RSDPVTLNVLY ++ 172 RTDSCSSAQAQY + 174 SADDIRGIQSLY +++ +++ + +++ 175 SDVTPLTF +++ ++ 176 SRTINVSNLY + + 177 SSDEVNFLVY + + 178 SSDSSTLPKL + 179 STAKSATWTY ++ 183 TLEDIATSHLY + 185 TSDSNLNKY ++ 188 VSDAKLDKY + 189 VSDSECLSRY +++ +++ 190 VTDGINPLIDRY ++ 192 VTEESFDSKFY + 193 VTEFSLNTY ++ + 194 VVADTKMIEY ++ + 195 VVDSVGGYLY + ++ 199 YLPQHTIETY + 200 YSDEDVTKY + ++ 201 YVGKEHMFY +++ +++ 202 KLAELEGALQK +++ + 203 KVKDTPGLGK ++ ++ + + 204 AVFDKFIRY ++ 205 SLDGAARPK + + ++ 206 KLIDLSQVMY + 207 RSFNGLLTMY + + 208 GLASRILDAK + + 209 RTQIPMSEK + 210 ATSGVPVYK + +

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211 TVNPVAIHK + 212 KAYEQVMHY + 213 LNINMTSPMGTK + 214 RTMSEAALVRK + 215 MMFSGPQILKL + 216 KLYAWELAF + 217 RILNQILYY + 218 KTLVAELLILK + + 219 RLRSSLVFK + 220 SPSVSQLSVL ++ +++ +++ 221 VPDVAQFVL +++ +++ 222 NPFYPEVEL +++ +++ 223 YPKDIYSSF ++ +++ 224 GPQPWHAAL +++ ++ + 225 LPFDGPGGIL +++ ++ 226 SPRMSGLLSQT +++ 227 YPRGNHWAVGH +++ 228 YPRGNHWAVGHL +++ 229 VPLPAGGGTV +++ 230 VPLPAGGGTVL +++ 231 RPRALRDLQL + ++ + 232 RPRALRDLQLL + ++ + 233 KPYQGNPTF ++ 234 RAKNAGVTI ++ ++ 235 MPLKHYLLL ++ 236 RVRGGEDGDRAL ++ 237 RPAATAVISL ++ ++ 238 KPGPPWAAF ++ 239 YVPSASLFML + ++ 240 SPREVTVL ++ 241 SARLATDAL ++ 242 SPRWLPVSL ++ 243 RPIENRILIL + ++ 244 FPYVRDFVM ++ + 245 RIREHVPQL ++ + 246 TPLPAVIVL + ++ 247 RALLARLLLL ++ + 248 IPNWARQDL ++ + 249 VPSSRILQL ++ + 250 SPRDFLSGL ++ 251 VPRSSGQTV + + ++

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252 SPDIRNTTV ++ 253 RVIDAVRFTL ++ 254 NPFPHLITL + + + 255 MPLLENLYL + + 256 SPRVPSIEL + 257 LPRIPFADV + + ++ 258 LPRGPLASL + + 259 RPPAAGLRGISL + 260 YPQHPGLNA + + 261 APSARVGVC + + + 262 SAYPQRLEI + 263 HPAPYGDLL + + 264 RPILIIITL + 265 SPRQPPRLV + 266 HAYPPGPGL + 267 HPELVNHIVF + 268 YPLFRGINL + + 269 APRAPRLML + 270 APGPRFLVT + + 271 MPLPWSLALP + 272 MPLPWSLALPL + 273 MPLLWLRGF + + + 274 TPYQEHVAL + 275 APHPPLSW + 276 LPRAGGAFL + + 277 MPLFEPRVF + + + 278 HPMIDINGIIVF + + 279 SPARASPAL + 280 VPISEEGTPVL + 281 RPRAPVTPA + 282 MPQIETRVIL + + 283 RPHSLSSEL + + 284 FPVTSIFHTF + + 285 FPSFLTNSL + 286 VPTLRSEL + 287 APREEQQRSL + 288 FPQKFIDLL + 289 VPENHSVAL + 290 APYRPPDISL + 296 MPMQDIKM +++ +++ +++ 297 RAQLKLVAL +++

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298 FNKRKPLSL + ++ + 299 MAQFKEISL + ++ + 300 VASPKHCVL ++ ++ + + 301 YMHKLLLVL ++ 302 HLLQKQTSI ++ 303 LPFPKFTV ++ 304 ELKKLYCQI + 305 ALKLRVAVL + 306 ILKVKVGL + 307 ILLPRTVSL + + 308 MLKQKVEEL + 309 DAIQRKYSC + 310 LPPKKFVL + 311 EIRIRWQM + 312 EAMLRNKEL + 313 ELKKKEYEEL + 314 AIISRLVAL + 319 AEMLERVIKNY ++ +++ +++ 320 MEVDPIGHVYIF +++ +++ +++ 321 AEMLESVIKNY + +++ +++ 322 KEVDPAGHSY +++ +++ 323 SEFMQVIF +++ +++ 324 TDSIHAWTF +++ 325 QEQDVDLVQKY +++ +++ 326 QEMQHFLGL +++ +++ +++ 327 YEIEARNQVF +++ +++ +++ 328 FEYDFLLQRI ++ +++ +++ 329 NEHPSNNW ++ +++ 330 KEGDLGGKQW ++ ++ + + 331 EDAQGHIW ++ ++ 332 MEVPVIKI + ++ + ++ 333 AETLSTIQI ++ ++ + 334 AEDEPAAAHL ++ ++ + + 335 KELEATKQY ++ ++ + 336 ASSSGPMRWW ++ ++ 337 TENRYCVQL ++ 338 SEGSEPALLHSW ++ 339 SEPALLHSW ++ 340 TEFSLNTY ++ + 341 EEIEGKGSFTYF ++ 342 HEFSSPSHL ++

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343 I th I IVLY + 344 EEATGQFHVY + 345 IEFIHPQAF + + 346 VEAPGPVHVYW + + + 347 ALNPYQYQY + 348 AEIQGNINHV + 349 AEQDMRELTY + 350 GECDVFKEIL + 351 EEVNYINTF + 352 NEVLTYIKF + 353 GEIIMQNNW + 354 TEDPTILRI + 355 SDMVRFHLF + + + 356 EEGRVYLF + + 357 RELENCFQIQ + 358 KEADIHFLI + + + 359 DELFSIALY + 360 AEVPTTGVII + 361 SENLFASF + 362 SEKGVIQVY + 363 AELDKLTSV + 364 AETPIQNVI + 365 SEMNVNMKY + 366 AENLFRAF + + 367 GEVHPSEMI + + 368 GEFPVVRVQV + 369 EEIERFFKL + 370 YEDLSQKY + 371 GELALKKKI + 372 TEGIIMKDF + 373 MEMQKSPVF + 374 DEVNFLVY + + 375 VYSDLHAFYY ++ 376 KYVKDFHKF + 377 VYVGAVNRI + 378 KFLGPAEHLTF + 388 FSIPEGALVAV + 389 TLMEQPLTTL + 401 DDSDESYMEKSLY + 402 DTDSQRLAY + 405 ETEEGIYWRY ++

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409 FVDPLVTNY + 419 LSELKPMSY + 427 NSDEQKITEMVY + 429 NTEDSSMSGYLY + 432 NVDPVQHTY + 438 STDNFNCKY + + 442 TSDFSRFTNY + 450 YSDDGQKWTVY ++ 454 KVYTPSISK + 455 RIADIFVKK + + 456 SMFTAILKK + 457 SINKPTSER + 458 GIADFVLKY + 459 RPMQQARAQL +++ 460 MPMAGDMNGL ++ 461 RPILIIVTL + 462 RPFHTRATV ++ ++ + + 463 TPKAGPTL ++ ++ + + 464 YPRPGTPAA + 465 VPRPIFSQL + 466 APYKSVTSL + + 467 KPFSSFTSM + 468 SPMYGQAGL + 469 YPENGVVQM + 470 SPNSYFRVL + 471 KPRPDVTNEL + 472 NPRATDAQL + 476 AEEEEIMKKI +++ +++ + +++ 477 QENSYQSRL ++ +++ 478 SEIEQEIGSL ++ ++ 479 AEIQPQTQV + + + 480 GEVSGLTKDF + 481 RELQHEHSL + + 482 TEREWADEW + 483 EENDQSTHKW + 484 AEVGFVRFF + 485 SEIEDSTKQVF + 486 SEDDPILQI + 487 AEDQLHHSF + 488 TEFPIIKMY + 489 SEIGKAVGF +

The table shows peptides derived from genes that are highly overexpressed in lung cancer tissues (NSCLadeno = non-small cell lung carcinoma - adenocarcinoma; NSCLCflat = squamous non-small cell lung carcinoma; NSCLCother = non-small cell lung carcinoma of other subtypes; SCLC = small cell lung carcinoma) compared to a panel of normal tissues (+++), highly overexpressed in tumors compared to a panel of normal tissues (++) or overexpressed in tumors compared to a panel of normal tissues (+). The background level of this score was calculated from measurements of the following relevant normal tissues: adipose tissue, adrenal gland, bile ducts, blood cells, blood vessels, bone marrow, brain, cartilage, esophagus, eye, gallbladder, heart, head and neck, kidney, colon, liver, lungs, lymph node, nerve, parathyroid gland, pancreas, pituitary gland, pleura, skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, bladder, ureter. If data were available for several samples of the same type of fabric, the arithmetic mean of all corresponding samples was used for calculations.

Example 3. In vitro immunogenicity for MHC class I presented peptides.

To obtain information about the immunogenicity of the TUMAP peptides of the present invention, applicants conducted in vitro T cell priming studies based on repeated stimulation of CD8+ T cells with artificial antigen presenting cells (iAPCs) loaded with peptide-MHC complexes and anti-CD28 antibody. Thus, applicants could demonstrate immunogenicity for HLA-A*02:01, HLA-A*24:02, HLA-A*O1:O1, HLA-A*03:01, HLAB*07:02, HLA- B*O8:O1 and HLA-B*44:02 TUMAP peptides of the invention, demonstrating that these peptides are T cell epitopes against which human CD8+ T cell progenitors exist (Tables 10a and 10b).

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Priming of CD8+ T cells in vitro.

To perform in vitro stimulation with artificial antigen presenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28 antibody, we first isolated CD8+ T cells from fresh leukapheresis product HLA-A*02, HLA-A*24, HLA-A* 01, HLA-A*03, HLA-B*07, HLA-B*O8:O1 or HLA-B*44 by positive selection using CD8 microspheres (Miltenyi Biotec, Bergisch Gladbach, Germany) from healthy donors (after signing the form informed consent) from the University Hospital Mannheim, Germany.

PBMCs and isolated CD8+ lymphocytes were incubated prior to use in T cell medium (TCM) consisting of RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated human AB serum (PAN-Biotech, Eidenbach, Germany), 100 U /ml penicillin/100 μg/ml streptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro, Oberdorla, Germany) and 20 μg/ml gentamicin (Cambrex). 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma, Nuremberg, Germany) were also added to the SCI medium at this stage.

Preparation of pMHC and anti-CD28 antibody-coated microspheres, T cell stimulation, and readout were performed in a well-characterized in vitro system using four different pMHC molecules for each stimulation cycle and 8 different pMHC molecules for each readout cycle.

Purified mouse anti-human CD28 costimulatory IgG2a Ab 9.3 (Jung et al., 1987) was chemically biotinylated using sulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer (Perbio, Bonn, Germany). The microspheres used were 5.6 μm polystyrene particles coated with streptavidin (Bangs Laboratories, IL, USA).

pMHCs used for positive and negative control stimulations were A*02:01/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 532) from modified Melan-A/MART-1) and A*02:01/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO. 533), respectively.

800,000 microspheres/200 μl were added to the wells of a 96-well plate in the presence of 4 x 12.5 ng of another biotinylated pMHC complex, washed and then added with 600 ng of biotinylated anti-CD28 antibodies in a volume of 200 μl. Stimulation was performed in 96-well plates by coincubating 1 x 10 ⁶ CD8+ T cells with 2 x ^{10 5} washed coated microspheres in 200 μl TCM medium supplemented with 5 ng/ml IL-12 (PromoCell) for 3 days at 37°C. Half of the medium was then replaced with fresh TCM medium supplemented with 80 U/ml IL-2, and incubation was continued for 4 days at 37°C. This stimulation cycle was performed a total of three times. For reading from pMHC multimers, 8 different pMHC molecules per cycle were used. A two-dimensional combinatorial coding approach was used as previously described ( Andersen et al., 2012 ) with minor modifications relating to labeling with 5 different fluorochromes. Finally, multimer analysis was performed by staining cells with a near-infrared cell viability kit with Live/dead dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany), and pMHC multimers with fluorescent tags. A BD LSRII SORP cytometer equipped with suitable lasers and filters was used for analysis. Peptide-specific cells were counted as a percentage of all CD8+ cells. The results of multimer analysis were assessed using the FlowJo program (Tree Star, Oregon, USA). In vitro priming of specific multimer-positive CD8+ lymphocytes was assessed by comparison with negative control stimulations. Immunogenicity for a given antigen was determined if at least one evaluable in vitro stimulated well from a single healthy donor was found to contain a specific CD8+ T cell line after in vitro stimulation (i.e., when the well contained at least 1 % specific multimer-positive among CD8-positive T cells and the percentage of specific multimer-positive cells was at least 10 times higher than the median value of negative control stimulations).

In vitro immunogenicity for lung cancer peptides (including NSCLC and SCLC) For the analyzed peptides bound to HLA class I molecules, in vitro immunogenicity could be demonstrated by the generation of peptide-specific T cell lines. Typical flow cytometric analysis results following TUMAP-specific multimer staining for 16 peptides of the invention are shown in FIG. 3-11 along with appropriate negative controls. The results for 152 peptides according to the invention are summarized in table. 10a and 10b.

- 80 045010

Table 10a

In vitro immunogenicity of HLA class I peptides according to the invention

Seq ID No Subsequence Positive wells [%] 491 KYLEKYYNL ++ 492 NYEDHFPLL ++ 493 TYKYVDINTF + 494 RYLEKFYGL + 495 SYNDALLTF +++ 496 VFMKDGFFYF + 498 EYIRALQQL + 500 VWSDVTPLTF + 504 VYEKNGYIYF ++++ 510 KVLEHVVRV + 513 KLVELEHTL + 515 KIFEMLEGV + 516 YTFSGDVQL + 519 KIQEILTQV + 520 KIQEMQHFL + 525 RLDDLKMTV + 528 RLLDSVSRL +

Typical results of in vitro immunogenicity experiments conducted by the Applicant for the peptides of the invention. <20% = +; 20% - 49% = ++; 50% - 69% = +++; >= 70% = ++++.

Table 10b

In vitro immunogenicity of HLA class I peptides according to the invention

Seq ID No Subsequence Positive wells [%] HLA 136 ATDLVVLDRY It I It A*01 143 FIDYPKKEDY It | It A*01 153 KLDRSVFTAY ++++ A*01 160 LTEAVLNRY ll_|__|_lt A*01 164 NTDNLAVY II .11 A*01 173 RTEFNLNQY ++++ A*01 174 SADDIRGIQSLY It | It A*01 185 TSDSNLNKY II .11 A*01 187 VADLHLY ^++ + ¹¹ A*01 189 VSDSECLSRY ll_|__|_lt A*01 193 VTEFSLNTY II | II A*01 201 YVGKEHMFY It | It A*01 395 ASEASRLAHY ll_|__|_lt A*01 398 ASEQQALHTVQY II | II A*01 405 ETEEGIYWRY II | II A*01 430 NTEGLHHLY II | II A*01 436 QTETGTPYMLY II | II A*01 451 YSDSLVQKGY II | II A*01 452 YVDAVLGKGHQY IIill A*01 84 YVDINTFRL II | II A*02

- 81 045010

85 YIDEFQSLV ++ A*02 87 TLYPYQISQL ++ A*02 88 VQMVITEAQKV ++ A*02 89 ILSTTMVTV +++ A*02 93 TLQEKILQV II .11 A*02 94 VLPDIETLIGV +++ A*02 95 ITIGVLARV ++++ A*02 96 HLVGGLHTV +++ A*02 97 VLALVNSTV +++ A*02 101 FIISDWRFVL II | II A*02 125 VLQPFLPSI ++++ A*02 127 GLDGSLVFL ++ A*02 128 FLGTTPTL II | II A*02 129 VLYDKDAVYV ++ A*02 130 NLWGGQGLLGV II | II A*02 131 LLKEFVQRV ++++ A*02 132 ALWLVDPLTV +++ A*02 133 MTLPVDAVISV II | II A*02 392 SLFKDQMEL A*02 393 ILLPYLQTL II | II A*02 205 SLDGAARPK ++ A*03 208 GLASRILDAK II | II A*03 209 RTQIPMSEK II | II A*03 210 ATSGVPVYK ++++ A*03 214 RTMSEAALVRK II | II A*03 218 KTLVAELLILK II | II A*03 1 QYDPTPLTW II | II A*24 2 VWSNVTPLKF II | II A*24 4 SYEKVINYL II | II A*24 6 KYKDYFPVI II | II A*24 8 PFLPPAACFF II | II A*24 10 VWSDVTPLNF II | II A*24 eleven YYSKSVGFMQW ++ A*24 13 HYTYILEVF +++ A*24 15 KYALLLQDL +++ A*24 16 TYNPDFSSL II | II A*24 59 KYSTTFFMV ++ A*24 60 TYLSIFDQL II | II A*24 61 NYAENILTL ++ A*24 62 LYQEILAQL II | II A*24 65 VYPASKMFPFI II | II A*24 66 IYFRDSSFL ++ A*24

-82045010

72 RYEGILYTI A*24 76 WYGWHFPEL A*24 79 RYLADLPTL A*24 83 TYCQNIKEF A*24 375 VYSDLHAFYY A*24 379 NYIVPDKQIF A*24 380 VFQEKHHVI A*24 383 RYKQDVERF +++ A*24 384 KYVKVFDKF +++ A*24 386 VYNDHSIYVW ++ A*24 220 SPSVSQLSVL ++++ B*07 221 VPDVAQFVL ++ B*07 222 NPFYPEVEL II .11 B*07 223 YPKDIYSSF ++ B*07 224 GPQPWHAAL ++ B*07 225 LPFDGPGGIL ++++ B*07 226 SPRMSGLLSQT +++ B*07 228 YPRGNHWAVGHL ++ B*07 231 RPRALRDLQL ++ B*07 232 RPRALRDLQLL +++ B*07 233 KPYQGNPTF II | II B*07 237 RPAATAVISL II | II B*07 241 SARLATDAL +++ B*07 242 SPRWLPVSL ++++ B*07 244 FPYVRDFVM II | II B*07 245 RIREHVPQL ++ B*07 248 IPNWARQDL ++++ B*07 249 VPSSRILQL +++ B*07 250 SPRDFLSGL II | II B*07 252 SPDIRNTTV II | II B*07 274 TPYQEHVAL II | II B*07 285 FPSFLTNSL ++++ B*07 292 SPQRLRGLLL +++ B*07 293 RPRSALPRLLLP ++ B*07 294 GPTPNTGAAL +++ B*07 460 MPMAGDMNGL ++ B*07 462 RPFHTRATV ++ B*07 463 TPKAGPTL II | II B*07 473 LPRALLSSL ++ B*07 474 LPRLLPAL +++ B*07 320 MEVDPIGHVYIF II | II B*44 322 KEVDPAGHSY ++ B*44

-83 045010

323 SEFMQVIF II .11 B*44 325 QEQDVDLVQKY II | II B*44 326 QEMQHFLGL P_|_P B*44 328 FEYDFLLQRI II | II B*44 329 NEHPSNNW II | II B*44 330 KEGDLGGKQW II | II B*44 331 EDAQGHIW B*44 333 AETLSTIQI II | II B*44 334 AEDEPAAAHL II | II B*44 337 TENRYCVQL ++ B*44 338 SEGSEPALLHSW B*44 339 SEPALLHSW B*44 342 HEFSSPSHL II | II B*44 476 AEEEEIMKKI II | II B*44 477 QENSYQSRL II | II B*44 297 RAQLKLVAL II | II B*08 298 FNKRKPLSL II | II B*08 299 MAQFKEISL ++++ B*08 300 VASPKHCVL II | II B*08 303 LPFPKFTV ++ B*08 305 ALKLRVAVL II | II B*08 306 ILKVKVGL II | II B*08 307 ILLPRTVSL P_|_P B*08 308 MLKQKVEEL II | II B*08 311 EIRIRWQM II | II B*08 312 EAMLRNKEL II | II B*08 313 ELKKKEYEEL II | II B*08 314 AIISRLVAL ++ B*08 315 DIYQRALNL II | II B*08 316 VIKEKALTL II .11 B*08 318 EAAIRSVEL ++ B*08

Typical results of in vitro immunogenicity experiments performed by the Applicant for the peptides of the invention. <20% = +; 20-49% = ++; 50-69% = +++; >= 70% = ++++.

Example 4. Peptide synthesis.

All peptides were synthesized by standard and generally accepted solid-phase peptide synthesis using the Fmoc technique. The identity and purity of each individual peptide was determined using mass spectrometry and analytical RP HPLC. The peptides were obtained as a white or off-white lyophilisate (trifluoroacetate salt) with a purity of >50%. All TUMAP peptides are preferably administered as trifluoroacetate or acetate salts, and other salt forms are also possible.

Example 5 MHC Binding Assay.

The candidate T cell therapy peptides of the present invention were further tested for their ability to bind to MHC (affinity). Individual peptide-MHC molecule complexes were produced by a UV-induced ligand exchange reaction in which the UV-sensitive peptide is cleaved by UV radiation to produce an exchange product with the peptide of interest. Only candidate peptides that can effectively bind and stabilize peptide-responsive MHC molecules prevent dissociation of MHC complexes. To determine the yield of the exchange reaction product, ELISA analysis was performed based on the detection of the light chain (e2m) of stabilized complexes with MHC. This analysis was performed essentially as described by Rodenko et al. (Rodenko et al., 2006).

MAXISorp 96-well plates (NUNC) were supplemented with 2 μg/ml streptavidin in PBS overnight at room temperature, washed 4 times, and blocked for 1 h at 37°C in blocking buffer with 2% BSA. The resulting refolding monomers HLA-A*O2:O1/MLA-OO1 served as a standard covering the range of 15–500 ng/mL. Monomeric peptide-MHC complexes after the exchange reaction under the influence of UV radiation were diluted 100-fold in blocking buffer. Samples were incubated for 1 h at 37°C, washed four times, incubated for 1 h at 37°C with 2 μg/ml horseradish peroxidase conjugated to e2m antibody, washed again, and detected with TMB solution; the reaction was stopped with NH2SO4. The absorption value was measured

- 84 045010 at a wavelength of 450 nm. Candidate peptides that exhibit high turnover yield (preferably greater than 50%, most preferably greater than 75%) are generally preferred for the generation and production of antibodies or fragments thereof and/or T cell receptors or fragments thereof, as they exhibit sufficient avidity in relation to MHC molecules and prevent the dissociation of MHC complexes.

Table 11

Indicators of binding to MHC class I molecules

Seq ID No Subsequence Peptide exchange 134 AAEIGDKSWLY ++++ 135 ASEDSVLLY ++++ 136 ATDLVVLDRY ++++ 137 ATSKFMEFY ++++ 138 DSDSCHFNY ++++ 139 ECDMAFHIY ++++ 140 ESDREELNY +++ 141 ESDVGVVVY +++ 142 EVAEPSVLFDLY +++ 143 FIDYPKKEDY +++ 144 FLDSQNLSAY +++ 145 FVDKPVAY +++ 146 GLNTGSALSY ++ 147 GSSDSSTLPKL 148 GTEFTTILY ++++ 149 GTEFTTVLY ++++ 150 GTELLSLVY ++++ 151 HSDLKVGEY +++ 152 HTDSLHLLI +++ 153 KLDRSVFTAY +++ 154 LLDISQKNLY +++ 155 LLDPNPHMY ++++ 156 LLDSLREQY +++ 157 LMDRPIFY ++++ 158 LSDLLKQGY ++++ 159 LSDTSVIQFY ++++ 160 LTEAVLNRY +++ 161 LVDDGTHGQY +++ 162 LVDNSIRELQY +++ 163 NSDSSLTLREFY +++ 164 NTDNLAVY ++++ 165 NTDPTAPPY +++ 166 NTQITDIGRY +++ 167 QSDPGTSVLGY +++

-85 045010

168 QTDHPQPILDRY ++++ 169 RLDTPLYFSY ++++ 170 RSDDTAVYY ++++ 171 RSDPVTLNVLY ++++ 172 RTDSCSSAQAQY +++” 173 RTEFNLNQY ++++ 174 SADDIRGIQSLY ++++ 176 SRTINVSNLY +++” 177 SSDEVNFLVY ++++ 178 SSDSSTLPKL ++ 179 STAKSATWTY ++++ 180 STDPWIQMAY ++++ 181 TADGKTYYY +++” 182 TDYHVRVY +++” 183 TLEDIATSHLY ++++ 184 TSAHPEDSSFY +++” 185 TSDSNLNKY ++++ 186 TTDIIEKY ll^^^ll 187 VADLHLY +++” 188 VSDAKLDKY +++” 189 VSDSECLSRY ++++ 190 VTDGINPLIDRY +++” 191 VTDGSLYEGVAY ++++ 192 VTEESFDSKFY ++++ 193 VTEFSLNTY ++++ 194 VVADTKMIEY II .11 195 VVDSVGGYLY ++++ 196 WMFFVINY II | II 197 YADTVRPEFY +++” 198 YLDPVQRDLY ++++ 199 YLPQHTIETY ++ 200 YSDEDVTKY ++++ 201 YVGKEHMFY ++++ 394 ASEAEMRLFY ++++ 395 ASEASRLAHY ++++ 396 ASEFGNHYLY ++++ 397 ASEITSKGASLY ++++ 398 ASEQQALHTVQY ++++ 399 ATDIPCLLY ++++ 400 ATDISRQNEY +++” 401 DDSDESYMEKSLY ++++ 402 DTDSQRLAY +++”

- 86 045010

403 ELDSKVEVLTY +++ 404 ETARKFLYY ++++ 405 ETEEGIYWRY ++++ 406 ETEQTKFWDY ++++ 407 FSDNDKLYLY ++++ 408 FTEQWTDGY +++ 409 FVDPLVTNY ++++ 410 GSDHQSPSSSSSY ++++ 411 GTVYEDLRY ++++ 412 ILDEVIMGY ++++ 413 ISDRYYTALY ++++ 414 KTDESLTKY +++ 415 LLDPRSYHTY +++ 416 LLDTAQKNLY ++++ 417 LLEDKHFQSY ++++ 418 LSDPSGPKSY +++” 419 LSELKPMSY ++++ 420 LTEDKETLQY ++++ 421 LTELLERAAFY ++++ 422 MIDVTKSYY ++++ 423 NLDAVHDITVAY ++++ 424 NLDEEKQLLY +++” 425 NLDIIQQEY ++++ 426 NLDQATRVAY +++” 427 NSDEQKITEMVY ++++ 428 NSELSCQLY ++++ 429 NTEDSSMSGYLY ++++ 430 NTEGLHHLY ++++ 431 NTSDMMGRMSY ++++ 432 NVDPVQHTY +++” 433 QIDTGENLY ++++ 434 QTDCAPNNGY ++++ 435 QTDDTTWRTEY ++++ 436 QTETGTPYMLY ++++ 437 STDGKHWWEY ++++ 438 STDNFNCKY +++” 439 TLDAGKFQIY +++ 440 TLDENPGVRY +++ 441 TLDSALNAASYY ++++ 442 TSDFSRFTNY ++++ 443 TTDFPSESSFEY ++++ 444 TTDTVIRSY +++ 445 VLDQGKITEY +++ 446 VTAQVVGTERY ++++ 447 VVDEDHELIY +++” 448 YLDIPNPRY +++” 449 YLDRGTGNVSFY ++++ 450 YSDDGQKWTVY ++++ 451 YSDSLVQKGY ++++ 452 YVDAVLGKGHQY ++++

The binding of peptides restricted by HLA class I molecules to HLA-A*01 is distributed according to the yield of peptide exchange: >10% = +; >20% = ++; >50 = +++; > 75% = ++++.

- 87 045010

Table 12

Indicators of binding to MHC class I molecules

Seq ID No Subsequence Peptide exchange 84 YVDINTFRL ++++ 85 YIDEFQSLV ++++ 86 FVIDGFDEL ++++ 87 TLYPYQISQL ++++ 88 VQMVITEAQKV ++++ 89 ILSTTMVTV ++++ 91 FALPGLLHA +++ 92 NLRDLLSEV +++ 93 TLQEKILQV ++++ 94 VLPDIETLIGV ++++ 95 ITIGVLARV ++++ 96 HLVGGLHTV ++++ 97 VLALVNSTV ++++ 98 LQSSGLTLLL ++++ 99 FLKEKVPGI ++++ 100 RQYPTPFQL ++++ 101 FIISDWRFVL +++ 102 SLLEQAIAL ++++ 103 FLYYPDPVL ++++ 104 GMLDIFWGV ++++ 105 SLLTHIPTA ++++ 106 FIIDTTYPAYV ++++ 107 LLQGAIESV ++++ 109 LLLGSIGLLGV ++++ 110 LLADFQALL ++++ 111 ALCLLLHLL 112 SVSDGIHSV ++++ 113 AVLTGLVEV ++++ 114 ILDERQVLL ++++ 115 MLLETQDALYV ++++ 116 VLMEENSKL ++++ 117 FLDPNARPLV ++++ 118 ALSSVLHSI ++++ 119 RTADITVTV ++++ 120 ALLANLPAV ++++ 121 ALVDTLTGI ++++ 122 ALLEMFPEITV ++++ 123 LMAFFLAVV ++ 124 SVASVLLYL ++++ 125 VLQPFLPSI ++++ 126 FLSTVTSV ++++ 127 GLDGSLVFL ++++ 128 FLGTTPTL ++++ 129 VLYDKDAVYV ++++ 130 NLWGGQGLLGV ++++ 131 LLKEFVQRV ++++ 132 ALWLVDPLTV ++++ 133 MTLPVDAVISV ++++ 388 FSIPEGALVAV ++++ 389 TLMEQPLTTL ++++ 390 HIMPTVHTV ++++ 391 SLIDMRGIETV ++++ 392 SLFKDQMEL ++++ 393 ILLPYLQTL ++++

The binding of peptides restricted by HLA class I molecules to HLA-A*02 is distributed according to the yield of peptide exchange: >10% = +; >20% = ++; >50 = +++; > 75% = ++++.

- 88 045010

Table 13

Indicators of binding to MHC class I molecules

Seq ID No Subsequence Peptide exchange 202 KLAELEGALQK ++++ 203 KVKDTPGLGK ++++ 204 AVFDKFIRY ++++ 205 SLDGAARPK ++++ 206 KLIDLSQVMY ++++ 207 RSFNGLLTMY ++++ 208 GLASRILDAK ++++ 209 RTQIPMSEK ++++ 210 ATSGVPVYK ++++ 211 TVNPVAIHK ++++ 212 KAYEQVMHY ++++ 214 RTMSEAALVRK ++++ 215 MMFSGPQILKL ++++ 216 KLYAWELAF +++ 217 RILNQILYY ++++ 218 KTLVAELLILK +++ 219 RLRSSLVFK ++++ 453 AINTSIKNK ++++ 454 KVYTPSISK ++++ 455 RIADIFVKK ++++ 456 SMFTAILKK ++++ 457 SINKPTSER ++++ 458 GIADFVLKY ++++

The binding of peptides restricted by HLA class I molecules to HLA-A*03 is distributed according to the yield of peptide exchange: >10% = +; >20% = ++; >50 = +++; > 75% = ++++.

Table 14

Indicators of binding to MHC class I molecules

Seq ID No Subsequence Peptide exchange 1 QYDPTPLTW ++++ 2 VWSNVTPLKF ++++ 3 YLEKFYGL ++ 4 SYEKVINYL ++++ 5 RYMKKDYLI ++++ 6 KYKDYFPVI ++++ 7 VQQWSVAVF +++ 8 PFLPPAACFF ++++ 10 VWSDVTPLNF ++++ eleven YYSKSVGFMQW ++++ 12 STIRGELFFF 13 HYTYILEVF ++++ 14 SYSSCYSF +++ 15 KYALLLQDL ++++ 16 TYNPDFSSL +++ 17 YYADKKTFIVL +++ 18 DYIGSVEKW ++++ 19 ILKEDPFLF Hi Η 20 EFTTVLYNF ++++ 21 SYEVRSTF +++ 22 TQPGDWTLF ++++ 23 KFIISDWRF ++++

-89045010

- 90 045010

67 RYPGKFYRV ++++ 68 IYQQIIQTY ++++ 69 IMPEKFEFW ++++ 70 PYTNYTFDF ++++ 71 SYMVLAPVF ++++ 72 RYEGILYTI ++++ 73 SYIGLPLTL +++ 74 VYDQYFITL ++++ 75 NYIYSISVF ++++ 76 WYGWHFPEL +++ 77 AYTLLGHEFV 78 TWFPKTPMLF ++++ 79 RYLADLPTL ++++ 80 YYSPLRDLL ++++ 81 LYPEGLRLL ++++ 82 RFLPSPWI ++++ 83 TYCQNIKEF ++++ 375 VYSDLHAFYY ++++ 376 KYVKDFHKF ++++ 377 VYVGAVNRI ++++ 378 KFLGPAEHLTF ++++ 379 NYIVPDKQIF +++ 380 VFQEKHHVI +++ 381 TYSKKHFRI ++++ 382 IYHSHHPTL +++ 383 RYKQDVERF +++ 384 KYVKVFDKF ++++ 385 MYINEVERL ++++ 386 VYNDHSIYVW ++++ 387 RWLPQKNAAQF +++

The binding of peptides restricted by HLA class I molecules to HLA-A*24 is distributed according to the yield of peptide exchange: >10% = +; >20% = ++; >50 = +++; > 75% = ++++.

Table 15

Indicators of binding to MHC class I molecules

Seq ID No Subsequence Peptide exchange 220 SPSVSQLSVL ++++ 221 VPDVAQFVL ++++ 222 NPFYPEVEL +++ 223 YPKDIYSSF ++++ 224 GPQPWHAAL ++++ 225 LPFDGPGGIL ++++

- 91 045010

226 SPRMSGLLSQT ++++ 227 YPRGNHWAVGH ++++ 228 YPRGNHWAVGHL ++++ 229 VPLPAGGGTV +++ 230 VPLPAGGGTVL +++ 231 RPRALRDLQL ++++ 232 RPRALRDLQLL ++++ 233 KPYQGNPTF ++++ 234 RAKNAGVTI ++++ 235 MPLKHYLLL ++++ 236 RVRGGEDGDRAL ++++ 237 RPAATAVISL +++ 238 KPGPPWAAF ++++ 239 YVPSASLFML ++++ 240 SPREVTVL ++++ 241 SARLATDAL ++++ 242 SPRWLPVSL ++++ 243 RPIENRILIL ++++ 244 FPYVRDFVM ++++ 245 RIREHVPQL +++ 246 TPLPAVIVL ++++ 247 RALLARLLLL +++ 248 IPNWARQDL ++++ 249 VPSSRILQL ++++ 250 SPRDFLSGL ++++ 251 VPRSSGQTV ++++ 252 SPDIRNTTV ++++ 253 RVIDAVRFTL +++ 254 NPFPHLITL ++++ 255 MPLLENLYL ++++ 256 SPRVPSIEL ++++ 257 LPRIPFADV ++++ 258 LPRGPLASL ++++ 259 RPPAAGLRGISL ++++ 260 YPQHPGLNA +++ 261 APSARVGVC +++ 262 SAYPQRLEI II .11 263 HPAPYGDLL ++++ 265 SPRQPPRLV ++++ 267 HPELVNHIVF ++ 268 YPLFRGINL ++++ 269 APRAPRLML ++++

- 92 045010

270 APGPRFLVT ++++ 271 MPLPWSLALP +++ 272 MPLPWSLALPL ++++ 273 MPLLWLRGF ++ 274 TPYQEHVAL +++ 275 APHPPLSVV +++ 276 LPRAGGAFL +++ 278 HPMIDINGIIVF ++ 279 SPARASPAL +++ 280 VPISEEGTPVL +++ 281 RPRAPVTPA ++++ 282 MPQIETRVIL +++ 283 RPHSLSSEL ++++ 284 FPVTSIFHTF ++ 285 FPSFLTNSL ++++ 286 VPTLRSEL ++++ 287 APREEQQRSL +++ 288 FPQKFIDLL ++ 289 VPENHSVAL ++++ 290 APYRPPDISL ++++ 291 SPQRLRGLL ++++ 292 SPQRLRGLLL +++ 293 RPRSALPRLLLP ++++ 294 GPTPNTGAAL ++++ 295 KPEGTRIAV ++++ 459 RPMQQARAQL +++ 460 MPMAGDMNGL ++++ 462 RPFHTRATV ++++ 463 TPKAGPTL ++++ 464 YPRPGTPAA ++++ 465 VPRPIFSQL ++++ 466 APYKSVTSL ++++ 467 KPFSSFTSM ++++ 468 SPMYGQAGL ++++ 469 YPENGVVQM ++ 470 SPNSYFRVL ++++ 471 KPRPDVTNEL ++++ 472 NPRATDAQL ++++ 473 LPRALLSSL ++++ 474 LPRLLPAL ++++ 475 RPHKPGLYL ++++

The binding of peptides restricted by HLA class I molecules to HLA-B*07 is distributed according to the yield of peptide exchange: >10% = +; >20% = ++; >50 = +++; > 75% = ++++.

- 93 045010

Table 16

Indicators of binding to MHC class I molecules

Seq ID No Subsequence Peptide exchange 296 MPMQDIKM ++++ 297 RAQLKLVAL ++++ 298 FNKRKPLSL +++ 299 MAQFKEISL ++++ 300 VASPKHCVL +++ 301 YMHKLLLVL ++++ 302 HLLQKQTSI +++ 303 LPFPKFTV ++++ 304 ELKKLYCQI ++++ 305 ALKLRVAVL ++++ 306 ILKVKVGL ++++ 307 ILLPRTVSL ++++ 308 MLKQKVEEL ++++ 309 DAIQRKYSC +++ 310 LPPKKFVL ++++ 311 EIRIRWQM ++++ 312 EAMLRNKEL ++++ 313 ELKKKEYEEL ++++ 314 AIISRLVAL ++++ 315 DIYQRALNL ++++ 316 VIKEKALTL +++ 317 LVKVKVLL ++++ 318 EAAIRSVEL ++++

The binding of peptides restricted by HLA class I molecules to HLA-B*08 is distributed according to the yield of peptide exchange: >10% = +; >20% = ++; >50 = +++; > 75% = ++++.

Table 17

Indicators of binding to MHC class I molecules

Seq ID No Subsequence Peptide exchange 319 AEMLERVIKNY ++++ 320 MEVDPIGHVYIF ++++ 321 AEMLESVIKNY +++ 322 KEVDPAGHSY ++ 323 SEFMQVIF +++ 324 TDSIHAWTF +++ 325 QEQDVDLVQKY ++ 326 QEMQHFLGL +++ 327 YEIEARNQVF +++

-94045010

328 FEYDFLLQRI ++++ 329 NEHPSNNW +++ 330 KEGDLGGKQW +++ 331 EDAQGHIW +++ 332 MEVPVIKI ++ 333 AETLSTIQI +++ 334 AEDEPAAAHL ++ 335 KELEATKQY ++ 336 ASSSGPMRWW ++ 337 TENRYCVQL ++++ 338 SEGSEPALLHSW +++ 339 SEPALLHSW +++ 340 TEFSLNTY - 341 EEIEGKGSFTYF +++ 342 HEFSSPSHL +++ 343 TEFTTVLY ++ 344 EEATGQFHVY +++ 345 IEFIHPQAF ++++ 346 VEAPGPVHVYW ++++ 347 ALNPYQYQY +++ 348 AEIQGNINHV +++ 349 AEQDMRELTY +++ 350 GECDVFKEIL +++ 351 EEVNYINTF +++ 352 NEVLTYIKF ++++ 353 GEIIMQNNW ++++ 354 TEDPTILRI +++ 355 SDMVRFHLF ++ 356 EEGRVYLF +++ 357 RELENCFQIQ +++ 358 KEADIHFLI ++++ 359 DELFSIALY ++++ 360 AEVPTTGVII +++ 361 SENLFASF ++++ 362 SEKGVIQVY +++ 363 AELDKLTSV +++ 364 AETPIQNVI +++ 365 SEMNVNMKY ++++ 366 AENLFRAF ++ 367 GEVHPSEMI +++ 368 GEFPVVRVQV ++ 369 EEIERFFKL +++

-95 045010

370 YEDLSQKY 371 GELALKKKI ++ 372 TEGIIMKDF ++ 373 MEMQKSPVF +++ 374 DEVNFLVY 476 AEEEEIMKKI ++ 477 QENSYQSRL +++ 478 SEIEQEIGSL +++ 479 AEIQPQTQV +++ 480 GEVSGLTKDF ++ 481 RELQHEHSL +++ 482 TEREWADEW +++ 483 EENDQSTHKW +++ 484 AEVGFVRFF ++++ 485 SEIEDSTKQVF +++ 486 SEDDPILQI +++ 487 AEDQLHHSF +++ 488 TEFPIIKMY ++ 489 SEIGKAVGF ++++

The binding of peptides restricted by HLA class I molecules to HLA-B*44 is distributed according to the yield of peptide exchange: >10% = +; >20% = ++; >50 = +++; > 75% = ++++.

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Claims (13)

1. A peptide having a length of 10 amino acids and consisting of the amino acid sequence SEQ ID No. 220, or a pharmaceutically acceptable salt thereof. 2. The peptide according to claim 1, wherein said peptide has the ability to bind to an MHC class I or II molecule and wherein said peptide, when bound to said MHC, is able to be recognized by CD4 and/or CD 8 T cells. 3. A fusion protein comprising the peptide of any one of claims 1 and 2 and the N-terminal amino acids of the antigen-associated invariant chain (Ii) of HLA-DR. 4. An antibody or fragment thereof that specifically recognizes the peptide according to any of claims 1 and 2. 5. A T-cell receptor or fragment thereof that specifically reacts with an HLA ligand, said ligand being a peptide according to any one of claims 1 and 2. 6. The T-cell receptor according to claim 5, wherein said amino acid sequence of the ligand contains a peptide in accordance with any of claims 1 and 2. 7. The T cell receptor according to claim 6, wherein said T cell receptor is present in the form of a soluble molecule. 8. A method for producing activated T lymphocytes in vitro, where the method includes contacting the T cells in vitro with antigen-loaded human MHC class I or II molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct simulating the antigen-presenting cell for a period of time, sufficient to activate said T cells in an antigen-specific manner, wherein said antigen is a peptide according to any one of claims 1 and 2. 9. A pharmaceutical composition containing at least one active ingredient selected from the group consisting of a peptide according to any one of claims 1 and 2, a fusion protein according to claim 3, an antibody or fragment thereof according to claim 4, a T-cell receptor or a fragment thereof according to claims 5-7 or an activated T lymphocyte obtained by the method according to claim 8, and a pharmaceutically acceptable carrier. 10. Use of the peptide according to any one of claims 1 and 2 for the manufacture of a medicinal product for the treatment of cancer. 11. Use of an antibody or a fragment thereof according to claim 4 for the manufacture of a medicinal product for the treatment of cancer. 12. Use of a T-cell receptor or a fragment thereof according to claims 5-7 for the manufacture of a medicinal product for the treatment of cancer. 13. Use of the activated T lymphocyte obtained by the method according to claim 8 for the manufacture of a medicinal product for the treatment of cancer.